Methods of identifying compounds that modulate il-4 receptor-mediated ige synthesis utilizing a chloride intracellular channel 1 protein

ABSTRACT

The present provides compounds capable of modulating IL-4 receptor-mediated IgE production, as well as IL-4 induced processes associated therewith, methods and kits for identifying such compounds that utilize a chloride intracellular channel 1 (CLIC1) as a surrogate analyte and methods of using the compounds in a variety of in vitro, in vitro and ex vivo contexts.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/197,945, filed Jul. 16, 2002, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to compounds that modulate processesassociated with isotype switching of B cells and IgE production, methodsand kits for identifying such compounds and methods of using suchcompounds in a variety of contexts, such as for the treatment orprevention of diseases associated with or characterized by productionand/or accumulation of IgE, including anaphylactic hypersensitivity orallergic reactions, allergic rhinitis, allergic conjunctivitis, systemicmastocytosis, hyper IgE syndrome, and IgE gammopathies, atopic disorderssuch as atopic dermatitis, atopic eczema and atopic asthma, and B-celllymphoma

BACKGROUND OF THE INVENTION

The immune system protects the body against invasion by foreignenvironmental agents such as microorganisms or their products, foods,chemicals, drugs, molds, pollen, animal hair or dander, etc. The abilityof the immune system to protect the body against such foreign invadersmay be innate or acquired.

The acquired immune response, which stems from exposure to the foreigninvader, is extremely complex and involves numerous types of cells thatinteract with one another in myriad ways to express the full range ofimmune response. Two of these cell types come from a common lymphoidprecursor cell but differentiate along different developmental lines.One line matures in the thymus (T-cells); the other line matures in thebone marrow (B-cells). Although T- and B-cells differ in many functionalrespects, they share one of the important properties of the immuneresponse: they both exhibit specificity towards a foreign invader(antigen). Thus, the major recognition and reaction functions of theimmune response are contained within the lymph cells.

A third cell type that participates in the acquired immune response isthe class of cells referred to as antigen-presenting cells (APC). Unlikethe T- and B-cells, the APC do not have antigen-specificity. However,they play an important role in processing and presenting the antigen tothe T-cells.

While the T- and B-cells are both involved in acquired immunity, theyhave different functions. Both T- and B-cells have antigen-specificreceptors on their surfaces that, when bound by the antigen, activatethe cells to release various products. In the case of B-cells, thesurface receptors are immunoglobulins and the products released by theactivated B-cells are immunoglobulins that have the same specificity forthe antigen as the surface receptor immunoglobulins. In the case ofactivated T-cells, the products released are not the same as theirsurface receptor immunoglobulins, but are instead other molecules,called cytokines, that affect other cells and participate in theelimination of the antigen. One such cytokine, released by a class ofT-cells called helper T-cells, is interleukin-4 (IL-4).

The immunoglobulins produced and released by B-cells must bind to a vastarray of foreign invaders (antigens). All immunoglobulins share certaincommon structural features that enable them to: (1) recognize and bindspecifically to a unique structural feature on an antigen (termed anepitope); and (2) perform a common biological function after binding theantigen. Basically, each immunoglobulin consists of two identical light(L) chains and two identical heavy (H) chains. The H chains are linkedtogether via disulfide bridges. The portion of the immunoglobulin thatbinds the antigen includes the amino-terminal regions of both L and Hchains. There are five major classes of H chains, termed α, δ, ε, γ andμ, providing five different isotypes of immunoglobulins: IgA, IgD, IgE,IgG and IgM. Although all five classes of immunoglobulins may possessprecisely the same specificity for an antigen, they all have differentbiological functions.

While the immune system provides tremendous benefits in protecting thebody against foreign invaders, particularly those that cause infectiousdiseases, its effects are sometimes damaging. For example, in theprocess of eliminating an invading foreign substance some tissue damagemay occur, typically as a result of the accumulation of immunoglobulinswith non-specific effects. Such damage is generally temporary, ceasingonce the foreign invader has been eliminated. However, there areinstances, such as in the case of hypersensitivity or allergicreactions, where the immune response directed against even innocuousagents such as inhaled pollen, inhaled mold spores, insect biteproducts, medications and even foods, is so powerful that it results insevere pathological consequences or symptoms.

Such hypersensitivity or allergic reactions are divided into fourclasses, designated types I-IV. The symptoms of the type I allergicreactions, called anaphylactic reactions or anaphylaxis, include thecommon symptoms associated with mild allergies, such as runny nose,watery eyes, etc., as well as the more dangerous, and often fatal,symptoms of difficulty in breathing (asthma), asphyxiation (typicallydue to constriction of smooth muscle around the bronchi in the lungs)and a sharp drop in blood pressure. Also included within the class oftype I allergic reactions are atopic reactions, including atopicdermatitis, atopic eczema and atopic asthma.

Even when not lethal, such anaphylactic allergic reactions producesymptoms that interfere with the enjoyment of normal life. One need onlywitness the inability of an allergy sufferer to mow the lawn or hikethrough the woods to understand the disruptive force even mild allergieshave on everyday life. Thus, while the immune system is quitebeneficial, it would be desirable to be able to interrupt its responseto invading foreign agents that pose no risk or threat to the body.

IgE immunoglobulins are crucial immune mediators of such anaphylactichypersensitivity and allergic reactions, and have been shown to beresponsible for the induction and maintenance of anaphylactic allergicsymptoms. For example, anti-IgE antibodies have been shown to interferewith IgE function and alleviate allergic symptoms (Jardieu, 1995, Curr.Op. Immunol. 7:779-782; Shields et al., 1995, Int. Arch. AllergyImmunol. 107:308-312). Thus, release and/or accumulation of IgEimmunoglobulins are believed to play a crucial role in the anaphylacticallergic response to innocuous foreign invaders. Other diseasesassociated with or mediated by IgE production and/or accumulationinclude, but are not limited to, allergic rhinitis, allergicconjunctivitis, systemic mastocytosis, hyper IgE syndrome, IgEgammopathies and B-cell lymphoma.

Although IgEs are produced and released by B-cells, the cells must beactivated to do so (B-cells initially produce only IgD and IgM). Theisotype switching of B-cells to produce IgE is a complex process thatinvolves the replacement of certain immunoglobulin constant (C) regionswith other C regions that have biologically distinct effector functions,without altering the specificity of the immunoglobulin. For IgEswitching, a deletional rearrangement of the IgH chain gene locusoccurs, which results in the joining of the switch region of the μ gene,Sμ, with the corresponding region of the ε gene, Sε.

This IgE switching is induced in part by IL-4 (or IL-13) produced byT-cells. The IL-4 induction initiates transcription through the Sεregion, resulting in the synthesis of germline (or “sterile”) εtranscripts (that is, transcripts of the unrearranged Cε H genes) thatlead to the production of IgE instead of IgM.

IL-4 induced germline ε transcription and consequent synthesis of IgE isinhibited by interferon gamma (IFN-γ), interferon alpha (IFN-α) andtumor growth factor beta (TGF-β). In addition to the IL-4 signal, asecond signal, also normally delivered by T-cells, is required forswitch recombination leading to the production of IgE. This secondT-cell signal may be replaced by monoclonal antibodies to CD40,infection by Epstein-Barr virus or hydrocortisone.

Generally, traditional treatments for diseases mediated by IgEproduction and/or accumulation regulate the immune system followingsynthesis of IgE. For example, traditional therapies for the treatmentof allergies include anti-histamines designed to modulate theIgE-mediated response resulting in mast cell degranulation. Drugs arealso known that generally downregulate IgE production or that inhibitswitching of, but not induction of, germline ε transcription (see, e.g.,Loh et al., 1996, J. Allerg. Clin. Immunol. 97(5):1141).

Although these treatments are often effective, treatments that act toreduce or eliminate IgE production altogether would be desirable. Byreducing or eliminating IgE production, the hypersensitivity or allergicresponse may be reduced or eliminated altogether. Accordingly, theavailability of compounds that are upstream modulators of IgEproduction, such as compounds capable of modulating, and in particularinhibiting, IL-4 receptor-mediated germline ε transcription, would behighly desirable.

The ability to screen for compounds capable of modulating IgEproduction, and in particular compounds that modulate IL-4 (or IL-13)induced germline ε transcription typically involves screening candidatecompounds in complex cell-based functional assays, such as thefunctional assays described in U.S. Pat. No. 5,958,707. These assaystypically involve contacting a cell comprising a reporter gene operablylinked to a promoter responsive to or inducible by IL-4 with a candidatecompound of interest in the presence of IL-4 and assessing the amount ofreporter gene product produced. The reporter gene is typically a genethat encodes a protein that produces an observable signal, such as afluorescent protein. The IL-4 inducible promoter may be a germline εpromoter. Compounds that antagonize (inhibit) IL-4 induced transcriptionwill yield reduced amounts of reporter gene product as compared tocontrol cells contacted with IL-4 alone. Compounds that agonize IL-4induced transcription will yield increased amounts of reporter geneproduct as compared to control cells contacted with IL-4 alone.Particularly useful functional assays for screening compounds for theability to modulate IL-4 inducible germline ε transcription aredescribed in U.S. Pat. No. 5,958,707, WO 99/58663 and WO 01/34806.

Although such functional screening assays are quite powerful andeffective, simpler assays that could be performed in cell-free systemsand/or that do not require a functional component, such as simplebinding assays with isolated proteins known to be involved in the IL-4signaling cascade responsible for the production of germline εtranscripts, and hence the production of IgE, would be beneficial.

SUMMARY OF THE INVENTION

These and other objects are furnished by the present invention, which inone aspect provides compounds, referred to herein as DL03 compounds,which are capable of modulating, and in particular inhibiting, the IL-4receptor-mediated signaling cascade involved in B-cell isotype switchingto, and consequent production of, IgE. The DL03 compounds of theinvention are generally 8 to 30 amino acid residue peptides or peptideanalogs, or pharmaceutically-acceptable salts thereof, characterized bystructural formula (I):Z¹-X¹˜X²˜X³˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸˜X⁹˜X¹⁰˜X¹¹˜X¹²˜X¹³˜X¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁸˜X¹⁹˜X²⁰-Z²

wherein:

-   -   X¹ is an aliphatic residue;    -   X² is an aromatic residue;    -   X³ is a small polar or small aliphatic residue;    -   X⁴ is a small polar or small aliphatic residue;    -   X⁵ is an aliphatic or nonpolar residue;    -   X⁶ is an aliphatic or nonpolar residue;    -   X⁷ is an aliphatic or nonpolar residue;    -   X⁸ is an aromatic residue;    -   X⁹ is small nonpolar residue;    -   X¹⁰ is an aliphatic or basic residue;    -   X¹¹ is a small polar or small aliphatic residue;    -   X¹² is a small polar or small aliphatic residue;    -   X¹³ is a small aliphatic or basic residue;    -   X¹⁴ is a small polar or small aliphatic residue;    -   X¹⁵ is a nonpolar or small aliphatic residue;    -   X¹⁶ is a small polar or small aliphatic residue;    -   X¹⁷ is a small aliphatic or basic residue;    -   X¹⁸ is a small aliphatic or conformationally-constrained        residue;    -   X¹⁹ is a small aliphatic or aliphatic residue; and    -   X²⁰ is a small aliphatic or basic residue;    -   Z¹ is RRN—, RC(O)NR—, RS(O)₂NR— or an amino-terminal blocking        group;    -   Z² is —C(O)OR, —C(O)O—, —C(O)NRR or a carboxyl-terminal blocking        group;    -   each R is independently selected from the group consisting of        hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,        arylalkyl, substituted arylalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl and substituted heteroarylalkyl;    -   each “˜” independently represents an amide, a substituted amide        or an isostere of an amide;    -   each “-” represents a bond, or a 1 to 10 residue peptide or        peptide analog; and    -   wherein one or more of X¹, X², X¹⁹, or X²⁰ may be absent.

The DL03 compounds of the invention preferably have between 10 and 25residues; more preferably, the DL03 compounds of the invention havebetween 12 and 20 residues. In particular, the more preferredembodiments have 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues.

Particular DL03 compounds of the invention include DL03wt(LYTSILLHGATTASMTKPLA (SEQ ID NO:1)), DL03IL (LYTSAALHGATTASMTKPLA (SEQID NO:2)), DL03KP (LYTSILLHGATTASMTAALA (SEQ ID NO:3)), DL03LA(LYTSILLHGATTASMTKPAR (SEQ ID NO:4)), DL03TS (LYAAILLHGATTASMTKPLA (SEQID NO:5)), DL03GA (LYTSILLHARTTASMTKPLA (SEQ ID NO:6)), DL03TT(LYTSILLHGAAAASMTKPLA (SEQ ID NO:7)), DL03AS (LYTSILLHGATTRAMTKPLA (SEQID NO:8)), DL03MT (LYTSILLHGATTASAAKPLA (SEQ ID NO:9)).

The DL03 compounds of the invention inhibit IL-4 (or IL-13) inducedgermline ε transcription in cellular assays. As a consequence of thisactivity, the DL03 compounds of the invention can be used to modulatethe IL-4 receptor-mediated signaling cascade involved in isotypeswitching to, and consequent production of, IgE. In a specificembodiment, the DL03 compounds may be used to inhibit IL-4 (or IL-13)induced IgE production as a therapeutic approach towards the treatmentor prevention of diseases associated with, characterized by or caused byIgE production and/or accumulation, such as anaphylactichypersensitivity or allergic reactions and/or symptoms associated withsuch reactions, allergic rhinitis, allergic conjunctivitis, systemicmastocytosis, hyper IgE syndrome, and IgE gammopathies, atopic disorderssuch as atopic dermatitis, atopic eczema and atopic asthma, and B-celllymphoma.

The DL03 compounds of the invention can also be used in assays toidentify other compounds capable of effecting the above activities, aswill be described in more detail, below.

In addition to their ability to inhibit IL-4 (or IL-13) induciblegermline ε transcription, and hence IL-4 receptor-mediated IgEproduction, the DL03 compound of the invention also bind chlorideintracellular channel 1 (CLIC1). In particular, three DL03 compounds ofthe invention, DL03 wt (LYTSILLHGATTASMTKPLA; SEQ ID NO:1), peptideDL03IL (LYTSAALHGATTASMTKPLA; SEQ ID NO:2), peptide DL03KP(LYTSILLHGATTASMTAALA; SEQ ID NO:3) and peptide DL03LA(LYTSILLHGATTASMTKPAR; SEQ ID NO: 4) were found to bind human CLIC1(hCLIC1) (NCBI Sequence Database NP_(—)001279.2 (SEQ: ID NO:12),corresponding nucleotide sequence at NM_(—)001288) in a yeast two hybridinteraction assay. Quite significantly, the ability of these DL03compounds to bind the hCLIC1 in this assay correlates with theirobserved ability to inhibit IL-4 (or IL-13) induced germline εtranscription. These observations provide the first evidence directlylinking CLIC1 to the IL-4 receptor-mediated signaling cascaderesponsible for isotype switching to, and consequent production of, IgE,and in particular as being an effector of germline ε transcription.Hence, these observations provide the first evidence directly linkingCLIC1 to the upstream regulation of isotype switching and/or IgEproduction.

This significant discovery enables the ability to use a CLIC1 as a“surrogate” analyte in screening assays to identify compounds thatmodulate or regulate the IL-4 receptor-mediated signaling cascadeleading to the production of IgE. Since induction of the ε promoter inresponse to IL-4 (or IL-13) is the first recognizable step necessary forisotype switching and consequent production of IgE, inhibition of IL-4(or IL-13) induced transcription of the ε promoter should preventB-cells from switching to and/or producing IgE. This significantdiscovery therefore permits the ability to use a CLIC1 as a surrogateanalyte in simple binding assays to identify compounds having a varietyof in vitro, in vivo and ex vivo therapeutic uses.

Accordingly, in another aspect, the invention provides methods ofidentifying compounds that modulate, and in particular inhibit, the IL-4receptor-mediated signaling cascade leading to the production of IgE.The methods generally comprise determining whether a candidate compoundof interest binds a CLIC1, wherein the ability to bind the CLIC1identifies the compound as being a modulator of the IL-4receptor-mediated signaling cascade leading to the production of IgE. Inone embodiment of the method, it is determined whether the candidatecompound competes for binding to the CLIC1 with a DL03 compound of theinvention, such as peptide DL03 wt (SEQ ID NO:1), peptide DL03IL (SEQ IDNO:2), peptide DL03KP (SEQ ID NO:3), or peptide DL03LA (SEQ ID NO:4). Ina specific embodiment of the method, compounds that inhibit IL-4 (orIL-13) induced germline ε transcription are identified. In a furtherembodiment, the methods comprise determining whether a candidatecompound of interest is a CLIC1 inhibitor.

In yet another aspect, the invention provides methods of identifyingcompounds that inhibit isotype switching of B-cells to produce IgEand/or IgE production. The methods generally comprise determiningwhether a candidate compound of interest binds a CLIC1, wherein theability to bind the CLIC1 identifies the compound as being an inhibitorof isotype switching and/or IgE production. In one embodiment of themethod, it is determined whether the candidate compound competes forbinding to the CLIC1 with a DL03 compound of the invention, such aspeptides DL03 wt (SEQ ID NO:1), DL03IL (SEQ ID NO:2), DL03KP (SEQ IDNO:3), DL03LA (SEQ ID NO:4).

In a specific embodiment of the method, compounds that inhibit IgEproduction are identified. In another specific embodiment, compoundsthat inhibit IL-4 receptor-mediated IgE production are identified. Instill another specific embodiment, compounds that inhibit CLIC1-mediatedIgE production are identified.

In still another aspect, the invention provides methods of identifyingcompounds that modulate, and in particular inhibit or downregulate,processes mediated by or associated with IL-4 receptor-mediated B-cellisotype switching and/or IgE production and/or accumulation. Suchprocesses include, but are not limited to, anaphylactic hypersensitivityor allergic reactions and/or symptoms associated with such reactions,allergic rhinitis, allergic conjunctivitis, systemic mastocytosis, hyperIgE syndrome, and IgE gammopathies, atopic disorders such as atopicdermatitis, atopic eczema and/or atopic asthma, and B-cell lymphoma. Themethods generally involve determining whether a candidate compound ofinterest binds a CLIC1, where the ability to bind the CLIC1 identifiesthe compound as being a modulator of a process mediated by or associatedwith IL-4 receptor-mediated isotype switching and/or IgE productionand/or accumulation. In one embodiment of the method, it is determinedwhether the candidate compound competes for binding the CLIC1 with aDL03 compound of the invention, such as peptide DL03 wt (SEQ ID NO:1),peptide DL03IL (SEQ ID NO:2), peptide DL03KP (SEQ ID NO:3), or peptideDL03LA (SEQ ID NO:4).

In yet another aspect, the invention provides methods of identifyingcompounds useful for treating disorders associated with, or mediated orcaused by, IgE production and/or accumulation. The methods generallycomprise determining whether a candidate compound of interest binds aCLIC1, wherein the ability to bind the CLIC1 identifies the compound asbeing useful for treating disorders associated with, or mediated orcaused by, IgE production and/or accumulation. In one embodiment of themethod, it is determined whether the candidate compound competes forbinding the CLIC1 with a DL03 compound of the invention, such as peptideDL03 wt (SEQ ID NO:1), peptide DL03IL (SEQ ID NO:2), peptide DL03KP (SEQID NO:3), or peptide DL03LA (SEQ ID NO:4). Diseases associated with, ormediated or caused by, IgE production and/or accumulation for whichtherapeutic compounds may be identified according to the methodsinclude, but are not limited to, anaphylactic hypersensitivity orallergic reactions and/or symptoms associated with such reactions,allergic rhinitis, allergic conjunctivitis, systemic mastocytosis, hyperIgE syndrome, and IgE gammopathies, atopic disorders such as atopicdermatitis, atopic eczema and atopic asthma, and B-cell lymphoma.

In another aspect, the invention provides compounds identified by thevarious screening methods of the invention.

In still another aspect, the invention provides pharmaceuticalcompositions. The compositions generally comprise a DL03 compound of theinvention, a compound that competes for binding a CLIC1 with a DL03compound of the invention or a compound identified by the screeningmethods of the invention and a pharmaceutically-acceptable carrier,excipient or diluent.

In yet another aspect, the invention provides methods of modulating, andin particular inhibiting or downregulating, the IL-4 receptor-mediatedsignaling cascade involved in B-cell isotype switching to produce,and/or consequent production of, IgE, or processes involved in thissignal transduction cascade, such as IL-4 (or IL-13) induced germline εtranscription. The method generally involves administering to a cell acompound that binds a CLIC1 in an amount effective to modulate this IL-4receptor-mediated signaling cascade. In one embodiment of the method,the compound inhibits IL-4 (or IL-13) induced germline ε transcription.In another specific embodiment, the compound inhibits CLIC1-mediatedgermline ε transcription. The method may be practiced in vitro, in vivoor ex vivo. In one embodiment, the cell is administered a DL03 compoundof the invention, such as peptide DL03 wt (SEQ ID NO:1), peptide DL03IL(SEQ ID NO:2), peptide DL03KP (SEQ ID NO:3), peptide DL03LA (SEQ IDNO:4), peptide DL03TS (SEQ ID NO:5), peptide DL03GA (SEQ ID NO:6),peptide DL03TT (SEQ ID NO:7), peptide DL03AS (SEQ ID NO:8), peptideDL03MT (SEQ ID NO:9) or a compound that competes for binding to a CLIC1with an active DL03 compound of the invention.

In yet another aspect, the invention provides methods of modulating, andin particular inhibiting or downregulating, isotype switching to IgEand/or IgE production. The method generally involves administering to acell an amount of a compound that binds a CLIC1 effective to modulateisotype switching to IgE and/or IgE production. The method may bepracticed in vitro, in vivo or ex vivo. In one embodiment, the cell isadministered a DL03 compound of the invention, such as peptide DL03 wt(SEQ ID NO:1), peptide DL03IL (SEQ ID NO:2), peptide DL03KP (SEQ IDNO:3), or peptide DL03LA (SEQ ID NO:4), peptide DL03TS (SEQ ID NO:5),peptide DL03GA (SEQ ID NO:6), peptide DL03TT (SEQ ID NO:7), peptideDL03AS (SEQ ID NO:8), peptide DL03MT (SEQ ID NO:9), or a compound thatcompetes for binding to a CLIC1 with an active DL03 compound of theinvention.

In still another aspect, the invention provides methods of treating orpreventing diseases associated with, or mediated or caused by, IgEproduction and/or accumulation. The method generally comprisesadministering to an animal suffering from such a disease an amount of acompound that binds a CLIC1 effective to treat or prevent the diseaseand/or one or more of its symptoms. In one embodiment, the compoundadministered is a DL03 compound of the invention, such as peptide DL03wt (SEQ ID NO:1), peptide DL03IL (SEQ ID NO:2), peptide DL03KP (SEQ IDNO:3), or peptide DL03LA (SEQ ID NO:4), peptide DL03TS (SEQ ID NO:5),peptide DL03GA (SEQ ID NO:6), peptide DL03TT (SEQ ID NO:7), peptideDL03AS (SEQ ID NO:8), peptide DL03MT (SEQ ID NO:9). In anotherembodiment, the compound administered is a compound that competes forbinding to a CLIC1 with an active DL03 compound of the invention.Diseases associated with, or mediated or caused by, IgE productionand/or accumulation that may be treated or prevented according to themethods of the invention include, but are not limited to, anaphylactichypersensitivity or allergic reactions (including food and drugallergies), and/or symptoms associated with such reactions, allergicrhinitis, allergic conjunctivitis, systemic mastocytosis, hyper IgEsyndrome, and IgE gammopathies, atopic disorders such as atopicdermatitis, atopic eczema and/or atopic asthma, and B-cell lymphoma. Themethod may be practiced therapeutically to treat the disease once theonset of the disease and/or its associated symptoms have alreadyoccurred, or prophylactically to prevent the onset of the disease and/orits associated symptoms. The methods may be practiced in veterinarycontexts or in the treatment of humans.

In a final aspect, the invention provides kits for carrying out thevarious methods of the invention. In one embodiment, the kit comprises aCLIC1 and a DL03 compound of the invention or a compound that competesfor binding the CLIC1 with a DL03 compound of the invention. The kit mayfurther include additional components for carrying out the methods ofthe invention, such as, by way of example and not limitation, buffers,labels and/or labeling reagents and/or instructions teaching methods ofusing the kits.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the nucleotide sequence of a 603 bp fragment of thehuman germline ε promoter (SEQ ID NO:11);

FIG. 2A provides a cartoon illustrating the diphtheria toxin (DT)selection of reporter cell line A5T4;

FIG. 2B provides a cartoon illustrating the tetracycline/doxycyclinecontrolled peptide expression system of reporter cell line A5T4;

FIG. 3 provides a cartoon illustrating the enrichment and screeningprocedure used to identify certain DL03 compounds of the invention;

FIG. 4 provides DNA transfer data for peptide DL03 wt;

FIG. 5A provides a cartoon outlining a yeast two hybrid (YTH) screeningassay used to identify potential binding partners or targets for peptideDL03 wt;

FIG. 5B provides a cartoon illustrating a yeast two hybrid (YTH) assayused to identify hCLIC1 as a binding partner or target for peptide DL03wt;

FIG. 6 provides a cartoon summarizing strategies for reconfirmingpotential targets identified in the YTH assay depicted in FIG. 5A;

FIG. 7 provides interaction graphic profiles for peptide DL03 wt andmutants derived therefrom;

FIG. 8 provides selection criteria for the interaction profiling methodused to confirm hCLIC1 as a binding partner for peptide DL03 wt;

FIG. 9 provides weighted graphic interaction/functional profiles forpeptide DL03 wt and mutants derived therefrom.

FIG. 10 illustrates the amino acid sequence for human CLIC1 (SEQ IDNO:10).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows: Three-Letter One-Letter Amino AcidAbbreviation Abbreviation Alanine Ala A Arginine Arg R Asparagine Asn NAspartate Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q GlycineGly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys KMethionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D,” the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the N->C direction inaccordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When specified on an individual basis, theone-letter abbreviation is preceded by either a “d” or an “r,” where “d”indicates the nucleoside is a 2′-deoxyribonucleoside and “r” indicatesthe nucleoside is a ribonucleoside. For example, “dA” designates2′-deoxyriboadenosine and “rA” designates riboadenosine. When specifiedon an aggregate basis, the particular nucleic acid or polynucleotide isidentified as being either an RNA molecule or a DNA molecule.Nucleotides are abbreviated by adding a “p” to represent each phosphate,as well as whether the phosphates are attached to the 3′-position or the5′-position of the sugar. Thus, 5′-nucleotides are abbreviated as “pN”and 3′-nucleotides are abbreviated as “Np,” where “N” represents A, G,C, T or U. When nucleic acid sequences are presented as a string ofone-letter abbreviations, the sequences are presented in the 5′->3′direction in accordance with common convention, and the phosphates arenot indicated.

Definitions

As used throughout the instant application, the following terms shallhave the following meanings:

“Promoter” or “Promoter Sequence” refers to a DNA regulatory regioncapable of initiating transcription of a downstream (3′ direction)coding sequence. A promoter typically includes a transcriptioninitiation site (conveniently defined, for example, by mapping withnuclease S1) and protein binding domains responsible for bindingproteins that initiate transcription.

“TGF-β Inducible Promoter” refers to a promoter that initiatestranscription when a cell comprising a nucleic acid molecule includingsuch a promoter is exposed to, or contacted with, TGF-β. While notintending to be bound by any particular theory of operation, it isbelieved that contacting a cell comprising such a promoter with TGF-βcauses the activation of a DNA-binding protein that then binds the TGF-βinducible promoter and induces transcription of coding sequencesdownstream of the promoter

An “α promoter” or a “germ line α promoter” is a TGF-β induciblepromoter that, when induced in a B-cell, leads to the production of IgAimmunoglobulins. Such germline α promoters are well-known in the art.Such promoters may be endogenous to a cell, or alternatively, they maybe engineered or exogenously supplied.

“IL-4 inducible promoter” refers to a promoter that initiatestranscription when a cell comprising a nucleic acid molecule includingsuch a promoter is exposed to, or contacted with, IL-4 or IL-13. Whilenot intending to be bound by any particular theory of operation, it isbelieved that contacting a cell comprising such a promoter with IL-4 (orIL-13) causes the activation of a DNA-binding protein that then bindsthe IL-4 inducible promoter and induces transcription of codingsequences downstream of the promoter.

An “ε promoter” or a “germline ε promoter” is an IL-4 inducible promoterthat, when induced in a B-cell, leads to the production of IgEimmunoglobulins. Such IL-4 inducible germline ε promoters are well-knownin the art. Such promoters may be endogenous to a cell or,alternatively, they may be engineered or supplied exogenously. Aspecific example of a germline ε promoter is the 603 bp IL-4 induciblefragment of the human ε promoter depicted in FIG. 1 (SEQ ID NO:11).

A compound that “modulates an IL-4 inducible germ line ε promoter” orthat “modulates IL-4 induced germ line ε transcription” has the abilityto change or alter expression downstream of the germline ε promoterinduced by IL-4 (or IL-13). The change in IL-4 induced downstreamexpression may occur at the mRNA (transcriptional) level or at theprotein (translational) level. Hence, the change in IL-4 induceddownstream expression may be monitored at the RNA level, for example byquantifying induced downstream transcription products, or at the proteinlevel, for example by quantifying the amount or activity of induceddownstream translation products.

The compound may act to modulate the IL-4 inducible germline ε promotervia any mechanism of action. For example, the compound may act tomodulate the IL-4 inducible germline ε promoter by interacting with orbinding a DNA binding protein involved in the IL-4 inducedtranscription, or by interacting with or binding the IL-4 induciblegermline ε promoter per se, or by interacting with or binding to amolecule that functions in the signalling cascade triggered by IL-4.

A compound that “modulates IL-4 receptor-mediated IgE production and/oraccumulation” has the ability to change or alter the amount of IgEproduced and/or accumulated by a B-cell activated via the IL-4 receptorwith IL-4 (or IL-13 or other IL-4 receptor ligand) and, in some cases, asecond signal known to cause, in combination with IL-4 (or IL-13),isotype switching of B-cells to produce IgE. Such second signal may be,for example, anti-CD40 monoclonal antibodies (anti-CD40 mAbs), infectionby Epstein-Barr virus or hydrocortisone. The compound may act tomodulate IL-4 receptor-mediated IgE production and/or accumulation viaany mechanism of action. For example, the compound may act to modulateIL-4 induced germline ε transcription, and hence isotype switching, orthe compound may act to modulate IgE production and/or accumulation inan already switched cell. An “IL-4 induced” activity includes activity(e.g., production of IgE, transcription of germline ε promoter isotypeswitching of B-cell) that is induced as a result of the binding of IL-4,IL-13 or other IL-4 receptor ligand to the IL-4 receptor.

“Identifying” in the context of screening assays means determiningwhether a candidate compound unknown to possess a particular property ofinterest possesses the property of interest, as well as confirming thata compound thought or known to possess a particular property of interestpossesses the property of interest.

Compounds that “compete for binding with a DL03 compound” compete forbinding to a CLIC1 (defined in a later section) with an active DL03compound of invention (described in more detail in a later section),such as peptide DL03 wt (SEQ ID NO:1), peptide DL03IL (SEQ ID NO:2),peptide DL03KP (SEQ ID NO:3), or peptide DL03LA (SEQ ID NO:4), or withanother compound that competes for binding to a CLIC1 with an activeDL03 compound of the invention. For example, if compound 1 competes forbinding to a CLIC1 with an active DL03 compound and a candidate compoundcompetes for binding to the CLIC1 with compound 1, then for purposes ofthe present invention, the candidate compound competes for binding witha DL03 compound. Where competition with a specific category of compoundis intended, the modifiers “directly” and “indirectly” are used, where“directly” refers to competition with the stated compound and“indirectly” refers to competition with another compound that itselfcompetes with the stated compound.

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic monovalenthydrocarbon group having the stated number of carbon atoms (i.e., C₁-C₆means from one to six carbon atoms) derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene oralkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Theterm “alkyl” is specifically intended to include groups having anydegree or level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds and groupshaving mixtures of single, double and triple carbon-carbon bonds. Wherea specific level of saturation is intended, the expressions “alkanyl,”“alkenyl,” and “alkynyl” are used. The expression “lower alkyl” refersto alkyl groups composed of from 1 to 6 carbon atoms.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl group. Typicalalkanyl groups include, but are not limited to, methanyl; ethanyl;propanyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butyanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl group having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkene. The groupmay be in either the cis or trans conformation about the double bond(s).Typical alkenyl groups include, but are not limited to, ethenyl;propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,cyclobuta-1,3-dien-1-yl, etc.; and the like.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl group having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkyne. Typicalalkynyl groups include, but are not limited to, ethynyl; propynyls suchas prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene, etc. Typicalparent aromatic ring systems include, but are not limited to,aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexylene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene, and the like.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonring atoms (i.e., C₅-C₁₄ means from 5 to 14 carbon ring atoms) derivedby the removal of one hydrogen atom from a single carbon atom of aparent aromatic ring system. Typical aryl groups include, but are notlimited to, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In preferredembodiments, the aryl group is (C₅-C₁₄) aryl, with (C₅-C₁₀) being evenmore preferred. Particularly preferred aryls are cyclopentadienyl,phenyl and naphthyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl and/orarylalkynyl is used. In preferred embodiments, the arylalkyl group is(C₆-C₁₆) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C₁-C₆) and the aryl moiety is (C₅-C₁₀). Inparticularly preferred embodiments the arylalkyl group is (C₆-C₁₃),e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is(C₁-C₃) and the aryl moiety is (C₅-C₁₀).

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms are each independently replacedwith the same or different heteroatoms or heteroatomic groups. Typicalheteroatoms or heteroatomic groups to replace the carbon atoms include,but are not limited to, N, NH, P, O, S, Si, etc. Specifically includedwithin the definition of “parent heteroaromatic ring systems” are fusedring systems in which one or more of the rings are aromatic and one ormore of the rings are saturated or unsaturated, such as, for example,arsindole, benzodioxan, benzofuran, chromane, chromene, indole,indoline, xanthene, etc. Also included in the definition of “parentheteroaromatic ring system” are those recognized rings that includesubstituents, such as benzopyrone. Typical parent heteroaromatic ringsystems include, but are not limited to, arsindole, benzodioxan,benzofuran, benzopyrone, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic group having the stated number of ring atoms(i.e., “5-14 membered” means from to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In preferred embodiments,the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 memberedheteroaryl being particularly preferred.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylakenyl and/or heterorylalkynylis used. In preferred embodiments, the heteroarylalkyl group is a 6-20membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moietyof the heteroarylalkyl is 1-6 membered and the heteroaryl moiety is a5-14-membered heteroaryl. In particularly preferred embodiments, theheteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety is 1-3 membered and the heteroaryl moiety is a5-10 membered heteroaryl.

“Substituted Alkyl, Aryl, Arylalkyl, Heteroaryl or Heteroarylakyl”refers to an alkyl, aryl, arylalkyl, heteroaryl or heteroarylakyl groupin which one or more hydrogen atoms is replaced with another substituentgroup. Exemplary substituent groups include, but are not limited to,—OR′, —SR′, —NR′R′, —NO₂, —NO, —CN, —CF₃, halogen (e.g., —F, —Cl, —Brand —I), —C(O)R′, —C(O)OR′, —C(O)NR′, —S(O)₂R′, —S(O)₂NR′R′, where eachR′ is independently selected from the group consisting of hydrogen and(C₁-C₆) alkyl.

The terms “percentage of sequence identity” and “percentage homology”are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage may becalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package),or by visual inspection (see generally, Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1995 Supplement) (Ausubel)). Examples of algorithms that are suitablefor determining percent sequence identity and sequence similarity arethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

While all of the above mentioned algorithms and programs are suitablefor a determination of sequence alignment and % sequence identity, fordetermination of % sequence identity in connection with the presentinvention, the BESTFIT or GAP programs in the GCG Wisconsin Softwarepackage (Accelrys, Madison Wis.), using default parameters provided, arepreferred.

The DL03 Compounds

The DL03 compounds of the invention are generally peptides and/orpeptides analogs which, as will be discussed in more detail below, arecapable of modulating a variety of processes involved in IL-4receptor-mediated isotype switching of B-cells to produce IgE. The DL03compounds of the invention are generally peptides or peptide analogs, orpharmaceutically-acceptable salts thereof, that are from 8 to 30residues in length and include a “core” peptide or peptide analog,comprising at least 8 consecutive amino acid residues, preferably atleast 10 consecutive amino acid residues, more preferably at least 12amino acid residues, according to structural formula (II):X¹˜X²˜X³˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸˜X⁹˜X¹⁰˜X¹¹˜X¹²˜X¹³˜X¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁸˜X¹⁹˜X²⁰  (II)wherein:

-   -   X¹ is an aliphatic residue;    -   X² is an aromatic residue;    -   X³ is a small polar or small aliphatic residue;    -   X⁴ is a small polar or small aliphatic residue;    -   X⁵ is an aliphatic or nonpolar residue;    -   X⁶ is an aliphatic or nonpolar residue;    -   X⁷ is an aliphatic or nonpolar residue;    -   X⁸ is an aromatic residue;    -   X⁹ is a small nonpolar residue;    -   X¹⁰ is an aliphatic or basic residue;    -   X¹¹ is a small polar or small aliphatic residue;    -   X¹² is a small polar or small aliphatic residue;    -   X¹³ is a small aliphatic or basic residue;    -   X¹⁴ is a small polar or small aliphatic residue;    -   X¹⁵ is a nonpolar or small aliphatic residue;    -   X¹⁶ is a small polar or small aliphatic residue;    -   X¹⁷ is a small aliphatic or basic residue;    -   X¹⁸ is a small aliphatic or conformationally-constrained        residue;    -   X¹⁹ is a small aliphatic or aliphatic residue; and    -   X²⁰ is a small aliphatic or basic residue.

The DL03 compounds of the invention include linear, branched and cyclicpeptides and peptide analogs.

The DL03 compounds of the invention and/or the “core” peptides orpeptide analogs of structure (II) are defined, in part, in terms ofamino acids or residues bearing side chains belonging to certaindesignated classes. The definitions of the various classes of aminoacids or residues that define structure (II), and hence the DL03compounds of the invention, are as follows:

“Hydrophilic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-His (H), L-Arg (R) and L-Lys (K).

“Polar Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

“Hydrophobic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic Amino Acid or Residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. The aromatic or heteroaromatic ring maycontain one or more substituents such as —OH, —OR″, —SH, —SR″, —CN,halogen (e.g., —F, —Cl, —Br, —I), —NO₂, —NO, —NH₂, —NHR″, —NR″R″,—C(O)R″, —C(O)O—, —C(O)OH, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)NR″R″ andthe like, where each R″ is independently (C₁-C₆) alkyl, substituted(C₁-C₆) alkyl, (C₂-C₆) alkenyl, substituted (C₂-C₆) alkenyl, (C₂-C₆)alkynyl, substituted (C₂-C₆) alkynyl, (C₅-C₁₀) aryl, substituted(C₅-C₁₀) aryl, (C₆-C₁₆) arylalkyl, substituted (C₆-C₁₆) arylalkyl, 5-10membered heteroaryl, substituted 5-10 membered heteroaryl, 6-16 memberedheteroarylalkyl or substituted 6-16 membered heteroarylalkyl.Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y)and L-Trp (W). Although owing to the pKa of its heteroaromatic nitrogenatom L-His (H) is classified above as a basic residue, as its side chainincludes a heteroaromatic ring, it may also be classified as an aromaticresidue.

“Non-polar Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

The amino acid L-Cys (C) is unusual in that it can form disulfidebridges with other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present invention L-Cys (C) is categorized as apolar hydrophilic amino acid, notwithstanding the generalclassifications defined above.

The amino acid Gly (G) is also unusual in that it bears no side chain onits α-carbon and, as a consequence, contributes only a peptide bond to aparticular peptide sequence. Moreover, owing to the lack of a sidechain, it is the only genetically-encoded amino acid having an achiralα-carbon. Although Gly (G) exhibits a hydrophobicity of 0.48 accordingto the normalized consensus scale of Eisenberg (Eisenberg et al., 1984,supra), for purposes of the present invention, Gly is categorized as analiphatic amino acid or residue.

Owing in part to its conformationally constrained nature, the amino acidL-Pro (P) is also unusual. Although it is categorized herein as ahydrophobic amino acid or residue, it will typically occur in positionsnear the N- and/or C-termini so as not to deleteriously affect thestructure of the DL03 compounds. However, as will be appreciated byskilled artisans, DL03 compounds may include L-Pro (P) or other similar“conformationally constrained” residues at internal positions.

“Small Amino Acid or Residue” refers to an amino acid or residue havinga side chain that is composed of a total three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include Gly,L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) andL-Asp (D).

“Hydroxyl-containing residue” refers to an amino acid containing ahydroxyl (—OH) moiety. Genetically-encoded hydroxyl-containing aminoacids include L-Ser (S) L-Thr (T) and L-Tyr (Y).

As will be appreciated by those of skill in the art, the above-definedcategories are not mutually exclusive. Indeed, the delineated categoryof small amino acids includes amino acids from all of the otherdelineated categories except the aromatic category. Thus, amino acidshaving side chains exhibiting two or more physico-chemical propertiescan be included in multiple categories. As a specific example, aminoacid side chains having heteroaromatic moieties that include ionizableheteroatoms, such as His, may exhibit both aromatic properties and basicproperties, and can therefore be included in both the aromatic and basiccategories. The appropriate classification of any amino acid or residuewill be apparent to those of skill in the art, especially in light ofthe detailed disclosure provided herein.

While the above-defined categories have been exemplified in terms of thegenetically encoded amino acids, the DL03 compounds of the invention arenot restricted to the genetically encoded amino acids. Indeed, inaddition to the genetically encoded amino acids, the DL03 compounds ofthe invention may be comprised, either in whole or in part, ofnaturally-occurring and/or synthetic non-encoded amino acids. Certaincommonly encountered non-encoded amino acids of which the cycliccompounds of the invention may be comprised include, but are not limitedto: the D-enantiomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (NaI); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thecompounds of the invention may be comprised will be apparent to those ofskill in the art (see, e.g., the various amino acids provided in Fasman,1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRCPress, Boca Raton, Fla., at pp. 3-70 and the references cited therein,all of which are incorporated by reference). These amino acids may be ineither the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the DL03compounds of the invention. Non-limiting examples of such protectedamino acids, which in this case belong to the aromatic category, include(protecting groups listed in parentheses), but are not limited to:Arg(tos), Cys(methylbenzyl), Cys (nitropyridinesulfenyl),Glu(δ-benzylester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom),His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe DL03 compounds of the invention may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

The classifications of the genetically encoded and certain commonnon-encoded amino acids according to the categories defined above aresummarized in TABLE 1, below. It is to be understood that TABLE 1 is forillustrative purposes only and does not purport to be an exhaustive listof amino acids that can comprise the DL03 compounds of the invention.Other amino acids not specifically mentioned herein can be readilycategorized based on their observed physical and chemical properties inlight of the definitions provided herein. TABLE 1 Encoded and CertainCommon Non-Encoded Amino Acid Classifications Classifica- tion EncodedAmino Acids Non-encoded Amino Acids Hydrophobic Aromatic F, Y, W, H f,y, w, h, Phg, Nal, Thi, Tic, Pcf, Off, Mff, Pff, hPhe Non-Polar L, V, I,M, G, A, P l, v, i, m, g, a, p, Bua, Bug, MeIle, Nle, MeVal, Cha, MeGly,Aib Aliphatic A, V, L, I a, v, l, i, Dpr, Aib, Aha, MeGly, Bua, Bug,Mele, Cha, Nle, MeVal Hydrophilic Acidic D, E d, e Basic H, K, R h, k,r, Dpr, Orn, hArg, Paf, Dbu, Dab Polar C, Q, N, S, T c, q, n, s, t, Cit,AcLys, Mso, hSer Small G, A, V, C, N, S, g, a, v, c, n, s, t, d T, D

In the “core” peptides and peptide analogs of structure (II), the symbol“˜” between each specified residue X¹ designates a backbone constitutivelinking moiety. When the DL03 compounds of the invention are peptides,each “˜” between the various X^(n) represents an amide or peptidelinkage of the following polarity: —C(O)—NH—. It is to be understood,however, that the DL03 compounds of the invention include analogs ofpeptides in which one or more amide or peptide linkages are replacedwith a linkage other than an amide or peptide linkage, such as asubstituted amide linkage, an isostere of an amide linkage, or a peptidoor amide mimetic linkage. Thus, when used in connection with definingthe various X^(n) comprising the DL03 compounds of the invention, theterm “residue” refers to the C_(α) carbon and side chain moiety(ies) ofthe designated amino acid or class of amino acid. As a specific example,defining X¹ as being a “Gly residue” means that X¹ is C_(α)H₂. DefiningX¹ as being an “Ala residue” means that X¹ is C_(α)HCH₃ in which theC_(α) carbon is in either the D- or L-configuration. Defining X¹ asbeing an “A residue” means that X¹ is C_(α)HCH₃ in which the C_(α)carbon is in the L-configuration.

Substituted amide linkages that may be included in the DL03 compounds ofthe invention include, but are not limited to, groups of the formula—C(O)NR², where R² is (C₁-C₆) alkyl, (C₅-C₁₀) aryl, substituted (C₅-C₁₀)aryl, (C₆-C₁₆) arylalkyl, substituted (C₆-C₁₆) arylalkyl, 5-10 memberedheteroaryl, substituted 5-10 membered heteroaryl, 6-16 memberedheteroarylalkyl or substituted 6-16 membered heteroarylalkyl. In aspecific embodiment, R² is (C₁-C₆) alkanyl, (C₂-C₆) alkenyl, (C₂-C₆)alkynyl or phenyl.

Isosteres of amides that may be included in the DL03 compounds of theinvention generally include, but are not limited to, —NR³—SO—,—NR³—S(O)₂—, —CH₂—CH₂—, —CH═CH— (cis and trans), —CH₂—NH—, —CH₂—S—,—CH₂—O—, —C(O)—CH₂—, —CH(OH)—CH₂— and —CH₂—S(O)₂—, where R³ is hydrogenor R² and R² is as previously defined. These interlinkages may beincluded in the DL03 compounds of the invention in either the depictedpolarity or in the reverse polarity. Peptide analogs including suchnon-amide linkages, as well as methods of synthesizing such analogs, arewell-known. See, for example, Spatola, 1983, “Peptide BackboneModifications,” In: Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins, Weinstein, Ed., Marcel Dekker, New York, pp. 267-357(general review); Morley, 1980, Trends Pharm. Sci. 1:463-468; Hudson etal., 1979, Int. J. Prot. Res. 14:177-185 (—CH₂—NH —, —CH₂—CH₂); Spatolaet al, 1986, Life Sci. 38:1243-1249; Spatola, 1983, “Peptide BackboneModifications: the Ψ [CH₂S] Moiety as an Amide Bond Replacement,” In:Peptides: Structure and Function V, J. Hruby and D. H. Rich, Eds.,Pierce Chemical Co., Rockford, Ill., pp. 341-344 (—CH₂—S—); Hann, 1982,J. Chem. Soc. Parkin Trans. I. 1:307-314 (—CH═CH—, cis and trans);Almquist et al., 1980, J. Med. Chem. 23:1392-1398 (—C(O)—CH₂—); EuropeanPatent Application EP 45665; Chemical Abstracts CA 97:39405(—CH(OH)—CH₂—); Holladay et al., 1983, Tetrahedron Lett. 24:4401-4404(—CH(OH)—CH₂—); and Hruby, 1982, Life Sci. 31:189-199 (—CH₂—S—).

Alternatively, one or more amide linkages may be replaced withpeptidomimetic and/or amide mimetic moieties. Non-limiting examples ofsuch moities are described in Olson et al., 1993, J. Med. Chem.36:3039-3049; Ripka & Rich, 1998, Curr. Opin. Chem. Biol. 2:441-452;Borchardt et al., 1997, Adv. Drug. Deliv. Rev. 27:235-256 and thevarious references cited therein.

While structure (II) contains 20 specified residue positions, it is tobe understood that the DL03 compounds of the invention may contain fewerthan 20 residues. Indeed, truncated forms of structure (II) containingas few as 8 residues that retain one or more of the utilities describedherein are considered to be within the scope of the present invention.Truncated forms of the compounds of structure (II) are obtained bydeleting one or more residues from either or both termini. Preferredtruncated forms of structure (II) will contain at least 10 residues;more preferred truncated forms of structure (II) will contain at least12 to at least 16 residues or more.

The core peptides or peptide analogs of structure (II) may also beextended at one or both termini. Typically, such extensions will rangefrom about 1 to about 5 residues, but may be even longer, so long as thecompound retains one or more of the utilities described herein. Forexample, one or both termini may be extended by 6, 7, 8, 9, 10 or evenmore residues.

In one embodiment of the invention, the extension has a sequence thatcorresponds to a sequence of a signal peptide capable of effectingtransport across membranes, such that the DL03 compound is a “fusionpolypeptide.” Such fusion polypeptides are particularly advantageous foradministering to cells DL03 compounds of the invention that may notreadily traverse cell membranes. The signal sequence may be fused toeither the N-terminal or C-terminal portion of the DL03 compound,depending upon the characteristics of the particular signal sequenceselected. Signal sequences capable of transporting molecules into cellsare well-known in the art. Any of these sequences may be used inconnection with the DL03 compounds of the invention. Specific examplesof such sequences include HIV Tat sequences (see, e.g., Fawell et al.,1994, Proc. Natl. Acad. Sci. USA 91:664; Frankel et al., 1988, Cell55:1189; Savion et al., 1981, J. Biol. Chem. 256:1149; Derossi et al.,1994, J. Biol. Chem. 269:10444; Baldin et al., 1990, EMBO J. 9:1511;U.S. Pat. No. 5,804,604; U.S. Pat. No. 5,670,617; and U.S. Pat. No.5,652,122, the disclosures of which are incorporated herein byreference), antennapedia sequences (see, e.g., Garcia-Echeverria et al.,2001, Bioorg. Med. Chem. Lett. 11:1363-1366; Prochiantz, 1999, Ann. NYAcad. Sci. 886:172-179; Prochiantz, 1996, Curr. Opin. Neurobiol.6:629-634; U.S. Pat. No. 6,080,724, and the references cited in all ofthe above, the disclosures of which are incorporated herein byreference) and poly(Arg) or poly(Lys) chains of 5-10 residues.Additional non-limiting examples of specific sequences can be found inU.S. Pat. No. 6,248,558; U.S. Pat. No. 6,043,339; U.S. Pat. No.5,807,746 U.S. Pat. No. 6,251,398; U.S. Pat. No. 6,184,038 and U.S. Pat.No. 6,017,735, the disclosures of which are incorporated herein byreference.

The terminus of the DL03 compounds of the invention corresponding to theamino terminus, if present, may be in the “free” form (e.g., H₂N—), oralternatively may be acylated with a group of the formula R²C(O)— orR²S(O)₂—, wherein R² is as previously defined. In one embodiment, R² isselected from the group consisting of (C₁-C₆) alkyl, (C₅-C₁₀) aryl,(C₆-C₁₆) arylalkyl, 5-10 membered heteroaryl or 6-16 memberedheteroarylalkyl. In a specific embodiment, the R² group is a group thatfacilitates entry of the DL03 compound into a cell. Such groups arewell-known in the art.

In another embodiment, the amino terminus may be “blocked” with ablocking group designed to impart the DL03 compound with specifiedproperties, such as a low antigenicity. Non-limiting examples of suchblocking groups include polyalkylene oxide polymers such as polyethyleneglycol (PEG). A variety of polymers useful for imparting compounds, andin particular peptides and proteins, with specified properties are knownin the art, as are chemistries suitable for attaching such polymers tothe compounds. Specific non-limiting examples may be found in U.S. Pat.Nos. 5,643,575; 5,730,990; 5,902,588; 5,919,455; 6,113,906; 6,153,655;and 6,177,087, the disclosures of which are incorporated herein byreference.

Of course, skilled artisans will appreciate that any of thesetransport-effecting, acylating and/or blocking groups may also beattached to a side chain moiety of a DL03 compound. Residues havingappropriate functionalities for attaching such groups will be apparentto those of skill in the art, and include, by way of example and notlimitation, Cys, Lys, Asp and Glu.

The terminus of the DL03 compounds corresponding to the C-terminus, ifpresent, may be in the form of an underivatized carboxyl group, eitheras the free acid or as a salt, such as a sodium, potassium, calcium,magnesium salt or other salt of an inorganic or organic ion, or may bein the form of a derivatized carboxyl, such as an ester, thioester oramide. Such derivatized forms of the compounds may be prepared byreacting a DL03 compound having a carboxyl terminus with an appropriatealcohol, thiol or amine. Suitable alcohols, thiols or amines include, byway of example and not limitation, alcohols of the formula R²OH, thiolsof the formula R²SH and amines of the formula R²NH₂, R²R²NH or NH₃,where each R² is, independently of the others, as previously defined.

The C-terminus may also include transport-effecting or other blockinggroups, such as those described above.

As will be recognized by skilled artisans, the various X^(n) residuescomprising the DL03 compounds of the invention may be in either the L-or D-configuration about their C_(α) carbons. In one embodiment, all ofthe C_(α) carbons of a particular DL03 compound are in the sameconfiguration. In some embodiments of the invention, the DL03 compoundscomprise specific chiralities about one or more C_(α) carbon(s) and/orinclude non-peptide linkages at specified locations so as to impart theDL03 compound with specified properties. For example, it is well-knownthat peptides composed in whole or in part of D-amino acids are moreresistant to proteases than their corresponding L-peptide counterparts.Thus, in one embodiment, the DL03 compounds are peptides composed inwhole or in part of D-amino acids. Alternatively, DL03 compounds havinggood stability against proteases may include peptide analogs includingpeptide linkages of reversed polarity at specified positions. Forexample, DL03 compounds having stability against tryptic-like proteasesinclude peptide analogs having peptide linkages of reversed polaritybefore each L-Arg or L-Lys residue; DL03 compounds having stabilityagainst chymotrypsin-like proteases include peptide analogs havingpeptide linkages of reversed polarity before each small and medium-sizedL-aliphatic residue or L-non-polar residue. In another embodiment, DL03compounds having stability against proteases include peptide analogscomposed wholly of peptide bonds of reversed polarity. Other embodimentshaving stability against proteases will be apparent to those of skill inthe art. Additional specific embodiments of the DL03 compounds of theinvention are described below.

The DL03 compounds of the invention can be in a linear form or a cyclicform, with or without branching. The cyclic forms can be cyclized viathe terminal groups or via side chain groups on internal or terminalresidues, through covalent or non-covalent linkages. Additional linkinggroups may also be present to facilitate cyclization.

In one specific embodiment, the DL03 compounds of the invention are20-residue peptides or peptide analogs according to structural formula(III):Z³-X¹˜X²˜X³˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸˜X⁹˜X¹⁰˜X¹¹˜X¹²˜X¹³˜X¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁸˜X¹⁹˜X²⁰˜-Z⁴  (III)wherein:

each X¹ through X²⁰ is as previously defined for structure (II);

Z³ is H₂N—, R⁴HN— or R⁴C(O)NH—;

Z⁴ is —C(O)O—, —C(O)OR⁴, —C(O)NHR⁴ or —C(O)NH₂;

each R⁴ is independently (C₁-C₆) alkyl or (C₁-C₆) alkanyl;

each “˜” is independently an amide linkage, a substituted amide linkageor an isostere of an amide linkage; and

each “-” represents a bond.

In another specific embodiment, the DL03 compounds of the invention arecompounds according to structural formula (III) in which each “˜” is anamide linkage.

In yet another specific embodiment, the DL03 compounds of the inventionare compounds according to structural formula (III) in which:

-   -   X¹ is L-Leu;    -   X² is L-Tyr;    -   X³ is a small polar or small aliphatic residue;    -   X⁴ is a small polar or small aliphatic residue;    -   X⁵ is an aliphatic or nonpolar residue;    -   X⁶ is an aliphatic or nonpolar residue;    -   X⁷ is an aliphatic or nonpolar residue;    -   X⁸ is a L-His;    -   X⁹ is a small nonpolar residue;    -   X¹⁰ is an aliphatic or basic residue;    -   X¹¹ is a small polar or small aliphatic residue;    -   X¹² is a small polar or small aliphatic residue;    -   X¹³ is a small aliphatic or basic residue;    -   X¹⁴ is a small polar or small aliphatic residue;    -   X¹⁵ is a nonpolar or small aliphatic residue;    -   X¹⁶ is a small polar or small aliphatic residue    -   X¹⁷ is a small aliphatic or a basic residue;    -   X¹⁸ is a small aliphatic or a conformationally-constrained        residue;    -   X¹⁹ is a small aliphatic or an aliphatic residue; and    -   X²⁰ is a small aliphatic or a basic residue.        Preferably, X³ is L-Thr or L-Ala, X⁴ is L-Ser or L-Ala, X⁵ is        L-Ile or L-Ala, X⁶ is L-Leu or L-Ala, X⁷ is L-Leu or L-Ala, X⁹        is L-Gly or L-Ala, X¹⁰ is L-Ala or L-Arg, X¹¹ is L-Thr or L-Ala,        X¹² is L-Thr or L-Ala, X¹³ is L-Ala or L-Arg, X¹⁴ is L-Ser or        L-Ala, X¹⁵ is L-Met or L-Ala, X¹⁶ is L-Thr or L-Ala, X¹⁷ is        L-Lys or L-Ala, X¹⁸ is L-Pro or L-Ala, X¹⁹ is L-Leu or L-Ala        and/or X²⁰ is L-Arg or L-Ala.

In another specific embodiment, the DL03 compounds of the inventioninclude compounds according to structural formula IV, and variants ofsuch compounds of formula IV in which 1, 2, 3, or 4, preferably 1 or 2,of the amino acid residues set forth in IV are replaced by another aminoacid selected from the same class (as described herein) as the originalamino acid or by an Ala or a Gly residue:Z¹-L˜Y˜T˜S˜I˜L˜L˜H˜G˜A˜T˜T˜A˜S˜M˜T˜K˜P˜L˜A-Z²  (IV)wherein “-”, “˜”, Z¹ and Z² are as defined previously for formula (I).

In still another specific embodiment, the DL03 compounds of theinvention are selected from the group consisting of DL03 wt(LYTSILLHGATTASMTKPLA(SEQ ID NO:1)), DL03IL (LYTSAALHGATTASMTKPLA (SEQID NO:2)), DL03KP (LYTSILLHGATTASMTAALA (SEQ ID NO:3)), DL03LA(LYTSILLHGATTASMTKPAR (SEQ ID NO: 4)), DL03TS (LYAAI LLHGATTASMTKPLA(SEQID NO:5)), DL03GA (LYTS ILLHARTTASMTKPLA (SEQ ID NO:6)), DL03TT (LYTS ILLHGAAAASMTKPLA (SEQ ID NO:7)) DL03AS (LYTS I LLHGATTRAMTKPLA (SEQ IDNO:8)), DL03MT (LYTS I LLHGATTASAAKPLA (SEQ ID NO:9)) and analogs andprotease-resistant analogs thereof.

Preferred DL03 compounds are selected from DL03 wt (SEQ ID NO:1), DL03IL(SEQ ID NO:2), DL03KP (SEQ ID NO:3)), DL03LA (SEQ ID NO: 4).

Active DL03 compounds of the invention are those that modulate, and inparticular inhibit or downregulate, IL-4 induced IgE production and/oraccumulation and/or processes associated therewith. The DL03 compoundsof the invention may be assessed for such activity in any standard assaythat assesses the ability of a compound to modulate IL-4 induced IgEproduction and/or accumulation. For example, a DL03 compound of theinvention may be administered to a human or animal B-cell (e.g., primaryB cells from blood, tonsils, spleens and other lymphoid tissues)stimulated with IL-4 (available from Pharmingen, Hamburg, Germany) andanti-CD40 mAbs (available from Ancell Corporation, Bayport Minn.) andthe amount of IgE produced measured, for example, by an ELISA technique,such as the ELISA technique described in Worm et al., 1998, Blood92:1713. The ELISA technique can use, for example, murine anti humanIgE, biotinylated anti human IgE and streptavidin biotinylatedhorseradish peroxidase complex. Specific ELISA assays and techniquesthat may be used are provided in the Examples section. Particular activeDL03 compounds include without limitation DL03 wt (SEQ ID NO:1), DL03IL(SEQ ID NO:2), DL03KP (SEQ ID NO:3), DL03LA (SEQ ID NO:4), DL03TS (SEQID NO:5), DL03GA (SEQ ID NO:6), DL03TT (SEQ ID NO:7) DL03AS (SEQ IDNO:8), DL03MT (SEQ ID NO:9).

For DL03 compounds that readily traverse cell membranes, the compoundmay be administered to the cell by contacting the cell with thecompound. DL03 compounds composed wholly of genetically-encoded aminoacids that do not readily traverse cell membranes may be administered tothe cell using well-known delivery techniques. In one embodiment, suchDL03 compounds may be administered using well-known retroviral vectorsand infection techniques pioneered by Richard Mulligan and DavidBaltimore with Psi-2 lines and analogous retroviral packaging systemsbased upon NIH 3T3 cells (see Mann et al., 1993, Cell 33:153-159, thedisclosure of which is incorporated herein by reference). Suchhelper-defective packaging cell lines are capable of producing all ofthe necessary trans proteins (gag, pol and env) required for packaging,processing, reverse transcribing and integrating genomes. Those RNAmolecules that have in cis the ψ packaging signal are packaged intomaturing retrovirions. Virtually any of the art-known retroviral vectorsand/or transfection systems may be used. Specific non-limiting examplesof suitable transfection systems include those described in WO 97/27213;WO 97/27212; Choate et al., 1996, Human Gene Therapy 7:2247-2253;Kinsella et al., 1996, Human Gene Therapy 7:1405-1413; Hofmann et al.,1996, Proc. Natl. Acac. Sci. USA 93:5185-5190; Kitamura et al., 1995,Proc. Natl. Acac. Sci. USA 92:9146-9150; WO 94/19478; Pear et al., 1993,Proc. Natl. Acac. Sci. USA 90:8392-8396; Mann et al., 1993, Cell33:153-159 and the references cited in all of the above, the disclosuresof which are incorporated herein by reference. Specific non-limitingexamples of suitable retroviral vector systems include vectors basedupon murine stem cell virus (MSCV) as described in Hawley et al., 1994,Gene Therapy 1: 136-138; vectors based upon a modified MFG virus asdescribed in Rivere et al., 1995, Genetics 92:6733; pBABE as describedin WO 97/27213 and WO 97/27212; and the vectors depicted in FIG. 11 ofWO 01/34806, the disclosures of which are incorporated herein byreference. Other suitable vectors and/or transfection techniques arediscussed in connection with gene therapy administration, infra.

A specific assay for assessing IL-4 induced IgE production that may beused to assay DL03 compounds of the invention is described in Worm etal, 2001, Int. Arch. Allergy Immunol. 124:233-236. Generally, a DL03compound modulates IL-4 induced IgE production if it yields an increaseor decrease in measured IgE levels of at least about 25% as compared tocontrol cells (i.e., cells activated with IL-4+ anti-CD40 Mabs but notexposed to the DL03 compound). DL03 compounds that increase IL-4 inducedIgE production are IgE agonists whereas DL03 compounds that decreaseIL-4 induced IgE production are IgE antagonists. Skilled artisans willappreciate that DL03 compounds that inhibit greater levels of IL-4induced IgE production, for example on the order of 50%, 60%, 70%, 80%,90%, or even more as compared to control cells, are particularlydesirable. Thus, while compounds that inhibit at least about 25% of IL-4induced IgE production as compared to control cells are active,compounds that inhibit at least about 50%, 75% or even more IL-4 inducedIgE production as compared to control cells are preferred.

In another embodiment, DL03 compounds may be assayed for the ability tomodulate IL-4 induced transcription of a germline ε promoter. Generally,such assays involve administering a DL03 compound to an IL-4 inducedcell comprising an IL-4 inducible germline ε promoter and assessing theamount of gene expression (i.e. transcription) downstream of the εpromoter. Depending upon the ability of the DL03 compound to traversecell membranes, it may be administered to the cell by contacting thecell with the compound or (for peptide compounds) via the retroviraltransfection techniques described supra. The amount of the downstreamgene expression may be assessed at the mRNA level, for example byquantifying the amount of a downstream transcription product produced,or at the translation level, for example by quantifying the amount of adownstream translation product produced. In one embodiment, the germlineε promoter is operably linked to a reporter gene that encodes a proteinthat produces an observable and/or detectable signal, such as afluorescent protein. Specific examples of suitable assays for assessingDL03 compounds for the ability to modulate germline ε transcription aredescribed in U.S. Pat. No. 5,958,707, WO 01/34806, WO 99/58663, commonlyowned copending application Ser. No. 09/712,821, filed Nov. 13, 2000 andcommonly owned copending application Ser. No. 09/076,624, filed May 12,1998, the disclosures of which are incorporated herein by reference.Generally, a DL03 compound modulates germline ε transcription if ityields an increase or decrease in measured downstream expression of atleast about 25% as compared to control cells activated with IL-4 but notexposed to the DL03 compound. DL03 compounds that increase downstreamexpression are IL-4 agonists, whereas compounds that decrease (i.e.inhibit) downstream expression are IL-4 antagonists. Skilled artisanswill appreciate that DL03 compounds that inhibit greater levels of IL-4induced germline ε transcription, for example on the order of 50%, 60%,70%, 80%, 90%, or even more as compared to control cells, areparticularly desirable. Thus, while compounds that inhibit at leastabout 25% of IL-4 induced germline ε transcription as compared tocontrol cells are active, compounds that inhibit at least about 50%, 75%or even more IL-4 induced germline ε transcription as compared tocontrol cells are preferred. In one embodiment of the invention, activeDL03 compounds are those that exhibit a reporter ratio of ≧1.1 in theA5T4 reporter line screening assay described in the examples section. Ingeneral, the “reporter ratio” is the ratio of the signal from a reporterunder control of the ε promoter in the absence of a DL03 compound tothat in the presence of the DL03 compound. In particular, for screeningin the A5T4 reporter line that has been transformed with the inhibitorpeptide vector, the reporter ratio can be determined from the ratio ofthe GFP fluorescence of IL-4 stimulated cells in the presence ofdoxycycline or tetracycline (i.e., when expression of the peptide isrepressed) to the GFP fluorescence of IL-4 stimulated cells in theabsence of doxycycline or tetracycline.

As mentioned previously, B-cells initially produce IgD and IgMimmunoglobulins and, when induced by the proper cytokines, produce IgEs.B-cells can be induced to produce other types of immunoglobulins, suchas IgGs and IgAs, as well. For example, in the presence of the cytokineinterleukin-2 (IL-2), B-cells produce IgG1; in the presence of acombination of IL-2 and TGF-β, B-cells produce IgA. In many situations,it is desirable to selectively modulate (increase or decrease) theproduction of a single immunoglobulin isotype, as such specificitypermits the ability to treat or prevent diseases associated with theproduction and/or accumulation of the specified immunoglobulin isotypewithout affecting the immune system generally. Thus, in one embodiment,the DL03 compounds specifically modulate IL-4 induced germline εtranscription or IL-4 induced IgE production and/or accumulation. By“specific” is meant that the DL03 compound modulates IL-4 induced IgEproduction and/or accumulation or IL-4 induced germline ε transcriptionbut does not significantly affect the production and/or accumulation ofanother immunoglobulin, or transcription of the promoter of another Igisotype. In a particular embodiment, an DL03 compound specificallyinhibits IL-4 induced germline transcription or IL-4 induced IgEproduction or accumulation. Such DL03 compound does not significantlyinhibit production and/or accumulation of another Ig isotype, ortranscription of another Ig isotype promoter, if the observed inhibitionof the other Ig isotype in an appropriate assay is on the order of 10%or less as compared to control cells. Such specificity may be withrespect to a single Ig isotype, or may be with respect to one or more Igisotypes. For example, a DL03 compound may be assessed for specificityby assaying its ability to inhibit, for example, IgA production and/oraccumulation or to inhibit germline α transcription in assays similar tothose described above, except that the cells are activated witheffectors suitable for IgA switching and synthesis and amount of IgAproduced or the amount of expression downstream of a germline α promoteris assessed. Specific, non-limiting examples of DL03 compounds thatspecifically inhibit IL-4 induced IgE production and/or IL-4 inducedgermline ε transcription include peptides DL03 wt (SEQ ID NO:1), DL03IL(SEQ ID NO:2), DL03KP (SEQ ID NO:3), DL03LA (SEQ ID NO:4) DL03 wt (SEQID NO:1), DL03IL (SEQ ID NO:2), DL03KP (SEQ ID NO:3), DL03LA (SEQ IDNO:4), DL03TS (SEQ ID NO:5), DL03GA (SEQ ID NO:6), DL03TT (SEQ ID NO:7)DL03AS (SEQ ID NO:8), and DL03MT (SEQ ID NO:9).

As will be discussed in more detail below, it has been discovered thatthe ability of certain DL03 compounds to inhibit IL-4 induced IgEproduction and/or IL-4 induced germline ε transcription is mediated bybinding a CLIC1. Accordingly, DL03 compounds may also be assessed foractivity based upon their ability to bind a CLIC1 using, for example,any of the CLIC1 binding assays described infra. Generally, active DL03compounds are those having a binding constant (Kd) on the order of 10 mMor less, with Kds in the range of 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1nM or even lower, being preferred. Alternatively, active DL03 compoundsare those that compete for binding a CLIC1 with another active DL03compound. In a specific embodiment, the DL03 compound competes forbinding a CLIC1 with peptides DL03 wt (SEQ ID NO:1), DL03IL (SEQ IDNO:2), DL03KP (SEQ ID NO:3), DL03LA (SEQ ID NO:4), DL03TS (SEQ ID NO:5),DL03GA (SEQ ID NO:6), DL03TT (SEQ ID NO:7) DL03AS (SEQ ID NO:8), orDL03MT (SEQ ID NO:9). The ability of a DL03 compound to compete forbinding a CLIC1 with another DL03 compound may be assessed usingconventional competitive binding assay techniques. Generally, activeDL03 compounds are those that exhibit an IC₅₀ in the range of 1 mM orlower, with IC₅₀s in the range of 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1nM or even lower, in such competitive binding assays being preferred.

Chemical Synthesis of the DL03 Compounds

DL03 compounds of the invention may be prepared using standardtechniques of organic synthesis. DL03 compounds that are peptides may beprepared using conventional step-wise solution or solid phase synthesis(see, e.g., Chemical Approaches to the Synthesis of Peptides andProteins, Williams et al., Eds., 1997, CRC Press, Boca Raton Fla., andreferences cited therein; FMOC Solid Phase Peptide Synthesis: APractical Approach, Chan & White, Eds., 2000, IRL Press, Oxford,England, and references cited therein).

Alternatively, DL03 compounds, may be prepared by way of segmentcondensation, as described, for example, in Liu et al., 1996,Tetrahedron Lett. 37(7):933-936; Baca et al., 1995, J. Am. Chem. Soc.117:1881-1887; Tam et al., 1995, Int. J. Peptide Protein Res.45:209-216; Schnolzer and Kent, 1992, Science 256:221-225; Liu and Tam,1994, J. Am. Chem. Soc. 116(10):4149-4153; Liu and Tam, 1994, Proc.Natl. Acad. Sci. USA 91:6584-6588; Yamashiro and Li, 1988, Int. J.Peptide Protein Res. 31:322-334. The condensation technique isparticularly useful for synthesizing DL03 compounds comprising Glyresidues. Other methods useful for synthesizing the DL03 compounds ofthe invention are described in Nakagawa et al, 1985, J. Am. Chem. Soc.107:7087-7092. DL03 compounds that are peptide analogs may besynthesized using the various methods described in the references citedin connection with amide isosteres and amide and peptidomimetics, supra.

DL03 compounds containing N- and/or C-terminal blocking groups can beprepared using standard techniques of organic chemistry. For example,methods for acylating the N-terminus of a peptide or amidating oresterifying the C-terminus of a peptide are well-known in the art. Modesof carrying other modifications at the N- and/or C-terminus will beapparent to those of skill in the art, as will modes of protecting anyside-chain functionalities as may be necessary to attach terminalblocking groups.

Formation of disulfide linkages, if desired, is generally conducted inthe presence of mild oxidizing agents. Chemical oxidizing agents may beused, or the compounds may simply be exposed to atmospheric oxygen toeffect these linkages. Various methods are known in the art, includingthose described, for example, by Tam et al, 1979, Synthesis 955-957;Stewart et al., 1984, Solid Phase Peptide Synthesis, 2d Ed., PierceChemical Company Rockford, Ill.; Ahmed et al., 1975, J. Biol. Chem.250:8477-8482; and Pennington et al., 1991 Peptides 1990 164-166, Giraltand Andreu, Eds., ESCOM Leiden, The Netherlands. An additionalalternative is described by Kamber et al., 1980, Helv. Chim. Acta63:899-915. A method conducted on solid supports is described byAlbericio, 1985, Int. J. Peptide Protein Res. 26:92-97. Any of thesemethods may be used to form disulfide linkages in the DL03 compounds ofthe invention.

Cyclic peptides may be prepared or may result from the formation ofsingle or multiple disulfide bonds, other side-chains or head-to-tailcyclizations, either directly or by way of an optional linker. Thecyclic peptides may be prepared using any art-known techniques for thepreparation of cyclic peptides and cyclic peptide analogs. For example,the peptide or peptide analog may be prepared in linear or non-cyclizedform using conventional solution or solid phase peptide and/or peptideanalog syntheses and cyclized using standard chemistries. The linearpolypeptides can be cyclized with a linking group between the twotermini, between one terminus and the side chain of an amino acid in thepeptide or peptide derivative, or between the side chains to two aminoacids in the peptide or peptide derivative. Suitable procedures forsynthesizing the peptide and peptide analogs described herein, as wellas suitable chemistries for cyclizing such compounds, are well known inthe art. For references related to synthesis of cyclic peptides thereader is referred to Tam et al., 2000, Biopolymers 52:311-332; Camameroet al, 1998, Angew. Chem. Intl. Ed. 37: 347-349; Tam et al., 1998, Prot.Sci. 7:1583-1592; Jackson et al., 1995, J. Am. Chem. Soc. 117:819-820;Dong et al., 1995, J. Am. Chem. Soc. 117:2726-2731; Ishida et al., 1995,J. Org. Chem. 60:5374-5375; WO 95/33765, published Jun. 6, 1995; Xue andDeGrado, 1994, J. Org. Chem. 60(4):946-952; Jacquier et al., 1991, In:Peptides 1990 221-222, Giralt and Andreu, Eds., ESCOM Leiden, TheNetherlands; Schmidt and Neubert, 1991, In: Peptides 1990 214-215,Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands; Toniolo, 1990,Int. J. Peptide Protein Res. 35:287-300; Ulysse et al., 1995, J. Am.Chem. Soc. 117:8466-8467; Durr et al., 1991, Peptides 1990 216-218,Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands; Lender et al,1993, Int. J. Peptide Protein Res. 42:509-517; Boger and Yohannes, 1990,J. Org. Chem. 55:6000-6017; Brady et al., 1979, J. Org. Chem.4(18):3101-3105; Spatola et al., 1986, J. Am. Chem. Soc. 108:825-831;Seidel et al., 1991, In: Peptides 1990 236-237, Giralt and Andreu, Eds.,ESCOM Leiden, The Netherlands; Tanizawa et al., 1986, Chem. Phar, Bull.34(10):4001-4011; Goldenburg & Creighton, 1983, J. Mol. Biol.165:407-413; WO 92/00995 and WO 94/15958. These methods may be routinelyadapted to synthesize the cyclic compounds of the invention and areincorporated into this application by reference.

Recombinant Synthesis of the DL03 Compounds

If the DL03 compound is composed entirely of genetically-encoded aminoacids, or a portion of it is so composed, the peptide or the relevantportion may also be synthesized using conventional recombinant geneticengineering techniques.

For recombinant production, a polynucleotide sequence encoding thepeptide is inserted into an appropriate expression vehicle, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation.The expression vehicle is then transfected into a suitable target cellwhich will express the peptide. Depending on the expression system used,the expressed peptide is then isolated by procedures well-established inthe art. Methods for recombinant protein and peptide production are wellknown in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y. each of which is incorporated byreference herein in its entirety.)

To increase efficiency of production, the polynucleotide can be designedto encode multiple units of the peptide separated by enzymatic cleavagesites—either homopolymers (repeating peptide units) or heteropolymers(different peptides strung together) can be engineered in this way. Theresulting polypeptide can be cleaved (e.g., by treatment with theappropriate enzyme) in order to recover the peptide units. This canincrease the yield of peptides driven by a single promoter. In apreferred embodiment, a polycistronic polynucleotide can be designed sothat a single mRNA is transcribed which encodes multiple peptides (i.e.,homopolymers or heteropolymers) each coding region operatively linked toa cap-independent translation control sequence; e.g., an internalribosome entry site (IRES). When used in appropriate viral expressionsystems, the translation of each peptide encoded by the mRNA is directedinternally in the transcript; e.g., by the IRES. Thus, the polycistronicconstruct directs the transcription of a single, large polycistronicmRNA which, in turn, directs the translation of multiple, individualpeptides. This approach eliminates the production and enzymaticprocessing of polyproteins and may significantly increase yield ofpeptide driven by a single promoter.

Polynucleotides capable of generating or expressing certain cyclicpeptide embodiments of the compounds of the invention may be prepared invitro and/or in vivo. Polypeptides may be prepared from polynucleotidesto generate or express the cyclic peptides utilizing the trans splicingability of split inteins. Methods for making such polynucleotides toyield cyclic peptides are known in the art and are described, forexample, in WO 01/66565, WO 00/36093; U.S. Patent Application No.60/358,827, entitled “Cyclic Peptides and Analogs Useful to TreatAllergies”, filed on 21 Feb. 2002, the disclosures of which areincorporated herein by reference.

A variety of host-expression vector systems may be utilized to expressthe peptides described herein. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

The expression elements of the expression systems vary in their strengthand specificities. Depending on the host/vector system utilized, any ofa number of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5 K promoter) may beused; when generating cell lines that contain multiple copies ofexpression product, SV40-, BPV- and EBV-based vectors may be used withan appropriate selectable marker.

In cases where plant expression vectors are used, the expression ofsequences encoding the peptides of the invention may be driven by any ofa number of promoters. For example, viral promoters such as the 35S RNAand 19S RNA promoters of CaMV (Brisson et al., 1984, Nature310:511-514), or the coat protein promoter of TMV (Takamatsu et al.,1987, EMBO J. 6:307-311) may be used; alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.3:1671-1680; Broglie et al., 1984, Science 224:838-843) or heat shockpromoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986,Mol. Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plant virusvectors, direct DNA transformation, microinjection, electroporation,etc. For reviews of such techniques see, e.g., Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, N.Y., SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9.

In one insect expression system that may be used to produce the peptidesof the invention, Autographa californica, nuclear polyhidrosis virus(AcNPV) is used as a vector to express the foreign genes. The virusgrows in Spodoptera frugiperda cells. A coding sequence may be clonedinto non-essential regions (for example the polyhedron gene) of thevirus and placed under control of an AcNPV promoter (for example, thepolyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed (e.g., see Smith et al., 1983, J. Virol. 46:584; U.S.Pat. No. 4,215,051). Further examples of this expression system may befound in Current Protocols in Molecular Biology, Vol. 2, Ausubel et al.,eds., Greene Publish. Assoc. & Wiley Interscience.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promotermay be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicaliet al., 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).

Other expression systems for producing DL03 peptides of the inventionwill be apparent to those having skill in the art.

Purification of DL03 Compounds

The DL03 compounds of the invention can be purified by art-knowntechniques such as reverse phase chromatography high performance liquidchromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography and the like. The actual conditions used topurify a particular compound will depend, in part, on synthesis strategyand on factors such as net charge, hydrophobicity, hydrophilicity, etc.,and will be apparent to those having skill in the art.

For affinity chromatography purification, any antibody whichspecifically binds the compound may be used. For the production ofantibodies, various host animals, including but not limited to rabbits,mice, rats, etc., may be immunized by injection with a compound. Thecompound may be attached to a suitable carrier, such as BSA, by means ofa side chain functional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to a compound may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique originally described by Kohler & Milstein, 1975,Nature 256:495-497 and/or Kaprowski, U.S. Pat. No. 4,376,110; the humanB-cell hybridoma technique described by Kosbor et al., 1983, ImmunologyToday 4:72 and/or Cote et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030; and the EBV-hybridoma technique described by Cole et al.,1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96. In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454; Boss, U.S. Pat. No. 4,816,397; Cabilly, U.S.Pat. No. 4,816,567) by splicing the genes from a mouse antibody moleculeof appropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Or“humanized” antibodies can be prepared (see, e.g., Queen, U.S. Pat. No.5,585,089). Alternatively, techniques described for the production ofsingle chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can beadapted to produce compound-specific single chain antibodies.

Antibody fragments which contain deletions of specific binding sites maybe generated by known techniques. For example, such fragments includebut are not limited to F(ab′)2 fragments, which can be produced bypepsin digestion of the antibody molecule and Fab fragments, which canbe generated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for the peptide ofinterest.

The antibody or antibody fragment specific for the desired peptide canbe attached, for example, to agarose, and the antibody-agarose complexis used in immunochromatography to purify peptides of the invention.See, Scopes, 1984, Protein Purification: Principles and Practice,Springer-Verlag New York, Inc., N.Y., Livingstone, 1974, Methods InEnzymology: Immunoaffinity Chromatography of Proteins 34:723-731.

As will be recognized by skilled artisans, the above methods may also beused to prepare anti-CLIC1 antibodies. Such anti-CLIC1 antibodies may beused in the various methods described herein, for example, to inhibitIL-4 induced isotype switching and/or IgE production and/or to inhibitIL-4 induced germline ε transcription. Such antibodies may also be usedin the various therapeutic methods described herein.

Screening Assays

The present inventors have discovered that the IgE regulatory effects offour DL03 compounds of the invention, peptides DL03 wt (SEQ ID NO:1),DL03IL (SEQ ID NO:2), DL03KP (SEQ ID NO:3), and DL03LA (SEQ ID NO:4),are mediated by a Chloride intracellular channel 1 (CLIC1) (NCBISequence Database NP_(—)001279.2 (SEQ ID NO:12)). Specifically, thepresent inventors have discovered that these four DL03 compounds bind ahuman CLIC1 in yeast two hybrid (YTH) assays in a manner that correlateswith their ability to inhibit IL-4 induced germline ε transcription inin vitro cellular assays.

Chloride ion channels in plasma membranes play important roles in themaintaining cell volume, transepithelial transport, setting the membranepotential, bone resorption, and in the response to certainneurotransmitters (Jentsch, 1994, Curr. Opin. Cell Biol. 6, 600-601;Jentsch et al., 1997, Bioessays 19, 117-126). Chloride ion channels arealso present in intracellular membranes and play important roles inacidification of intracellular compartments and in exocytosis(Al-Awqati, 1995, Curr. Opin. Cell. Biol. 7, 504-508; 4. Redhead et al,1997, Mol. Biol. Cell 8, 691-704).

Four structurally unrelated types of chloride ion channels have beenidentified: 1.) the ligand-gated family (e.g. γ-aminobutyric acid andglycine receptors) (Hosie et al., 1997, Trends Neurosci. 20, 578-583);2.) the cystic fibrosis transmembrane conductance regulator (CFTR), amember of the ATP binding cassette family of proteins (Seibert et al.,1997, J. Bioenerg. Biomembr. 29, 429-442) 3.) the chloride ion channels(ClC) family (Jentsch et al., 1995, J. Physiol. (Lond.) 482, 19-25) and4.) the chloride intracellular channels (CLIC) family (Edwards, 1999,Am. J. Physiol. 276, F398-F408).

The CLIC family is defined by a COOH-terminal core segment of ˜230 aminoacids that is highly conserved among all family members. To date thereare at least seven members of the CLIC family: CLIC1 (NCC27) (Valenzuelaet al., 1997, J. Biol. Chem. 272, 12575-12582), CLIC2 (Heiss et al.,1997, Genomics 45, 224-228), CLIC3 (Qian et al., 1999, J. Biol. Chem.274, 1621-1627), CLIC4 (Duncan et al, 1997, J. Biol. Chem. 272,23880-23886), CLIC5 (Berryman et al., 2000, Mol. Biol. Cell 11,1509-1521), p64 (Landry et al., 1993, J. Biol. Chem. 268, 14948-14955),and parchorin (Nishizawa et al., 2000, J. Biol. Chem. 275, 11164-11173).

The first member of CLIC family described was the bovine intracellularchloride channel p64 (Landry et al. 1993, supra). A homologue of p64 wasisolated from rat brain tissue, and designated p64H1 (Duncan et al.1997, supra). CLIC1 isolated from the human monocyte cell line U937, isa much smaller protein than p64, containing only 241 amino acids. CLIC1shares 63% identity with bovine p64, 61% identity with human CLIC2, 66%with rat p64H1 and 49% identity with human CLIC3 (Valenzuela et al.,2000, J. Phys. 529.3, 541-552), 67% identity with human CLIC4 (NCBI:NP_(—)039234; gi: 7330335) and 63% identity with rat CLIC 5 (NCBI:NP_(—)446055.11 gi: 6758390).

The CLIC1 gene is highly conserved across species. A CLIC1 cDNA probecross hybridizes with genomic DNA from all the eukaryotic speciesstudied, including monkey, rat, mouse, dog, cow, rabbit and yeast. CLIC1shares 90-100% identity to a growing number of human, rat and mouseexpressed sequence tags (EST). CLIC1 is 99-100% identical over 109 aminoacids to a porcine EST (SSC24B10) and 63% identical over 152 amino acidsto an EST from zebrafish (AA497337).

The CLIC1 protein is a 27 kDa chloride ion channel that exists in cellsas a soluble cytoplasmic protein and an integral membrane protein. Inphospholipid membranes, CLIC1 functions as an anion selective channel(Tulk et al. (2002) Am. J. Physiol. 282(5):C1103-12). Within the CLICfamily, only CLIC1 and CLIC3 are dominantly nuclear in distribution(Qian et al., 1999, supra). The structure of the soluble form of CLIC1has been determined at 1,4-Å resolution (Harrop et al., 2001, J. Biol.Chem., Vol. 276, 48, 44993-45000). The soluble form of CLIC1 ismonomeric, although a recent publication (Warton et al. 2002 J. Biol.Chem. epublished Apr. 26, 2002) suggests that CLIC1 in ion channelsexists in a tetrameric assembly of subunits. CLIC1 is structurallyhomologous to the glutathione S-transferase superfamily, and it has aredox-active site resembling glutaredoxin (Harrop et al., 2001, supra).The structure of the complex of CLIC1 with glutathione shows thatglutathione occupies the redox-active site, which is adjacent to anopen, elongated slot lined by basic residues. The structure indicatesthat CLIC1 is likely to be controlled by redox-dependent processes(Harrop et al., 2001, supra).

Although the precise function of CLIC1 remains unclear, it has beenimplicated in the regulation of the cell cycle (Valenzuela et al., 2000,supra). Electrophysiological studies in Chinese hamster ovary (CHO-K1)cells indicated that CLIC1 chloride conductance varied according to thestage of the cell cycle, being expressed only on the plasma membrane ofcells in G2/M phase. (Valenzuela et al., 2000, supra). Cl⁻ ion channelblockers known to block CLIC1 led to arrest of CHO-K1 cells in the G2/Mstage of the cell cycle, the same stage at which this ion channel isselectively expressed on the plasma membrane (Valenzuela et al., 2000,supra). A protein homologous to CLIC1, DBP-1, was recently identified byits ability to bind to certain diarylsulfonylureas (DASUs)(US2002/0034764A1, EP0987552A2). As DASUs are known to inhibit theproduction of inflammatory cytokines like IL-1 and IL-18, DBP-1 has beenproposed as a target for screening compounds useful for treatment ofinflammatory disorders including asthma. However, the present inventorsare the first to discover a link between hCLIC1 and modulation of theIL-4 signaling cascade involved in the production of IgE, and inparticular to IL-4 induced isotype switching of B-cells to produce IgE.

This significant discovery enables, for the first time, the ability touse a CLIC1 as a “surrogate” analyte in simple binding assays to screenfor and/or identify compounds involved in IL-4 induced IgE regulation.Such compounds are useful in the treatment and/or prevention of diseasescaused by or associated with IgE production and/or accumulation, such asanaphylactic hypersensitivity or allergic reactions and or symptomsassociated with such reactions, allergic rhinitis, allergicconjunctivitis, systemic mastocytosis, hyper IgE syndrome and IgEgammopathies, and atopic disorders such as atopic dermatitis, atopiceczema and atopic asthma, and B-cell lymphoma.

Thus, the invention also provides methods and kits useful foridentifying compounds having specified utilities. In specificembodiments, the methods and kits may be used to identify compounds thatinhibit IL-4 induced IgE production and/or accumulation, compounds thatinhibit IL-4 induced isotype switching of B-cells to produce IgE,compounds that inhibit IL-4 induced germline ε transcription, and/orcompounds useful to treat or prevent diseases caused by or associatedwith IgE production and/or accumulation, such as those described above.

“Chloride intracellular channel 1” or “CLIC1” useful in the screeningmethods and kits of the invention include any protein recognized in theart as belonging to the CLIC1 family. In particular, useful CLIC1sinclude human CLIC1 (“hCLIC1”) (NP_(—)001279.2, XP_(—)004183.3, spO00299) shown in FIG. 10 as SEQ ID NO:10, and allelic and speciesvariants thereof, as well as fragments and fusions thereof that bind tothe DL03 compounds, particularly DL03 wt. Typically, such proteins willhave polypeptide sequences that share at least about 80% identity at theamino acid level with hCLIC1. Preferably, the CLIC1 protein employed inthe methods of the present invention will have at least 85%, 90%, 95% oreven higher % identity with hCLIC1. Specific examples of CLIC1s suitablefor use in the methods and kits of the invention include CLIC1 derivedfrom humans (Valenzuela et al., 1997, supra) as well as the variouscorresponding mammalian homologs thereof (for example, mouse, rabbit,ect.). The amino acid sequences of these various CLIC1s, as well as thesequences of nucleic acid molecules encoding these CLIC1s, are known inthe art and can be found in the following references and/or NCBI(GenBank) entries: Mus musculus CLCP (gi|3986758|gb|AAC84155.1;Oryctolagus cuniculus chloride intracellular channel protein(gi|14572050|gb|AAK67356.1|AF387765_(—)1).

As will be recognized by skilled artisans, mutants and/or fragments of aCLIC1 may also be used in the assays and kits of the invention. Mutantsand/or fragments of CLIC1 that are useful in this regard are thosemutants and/or fragments that retain the ability to bind an active DL03compound, preferably peptide DL03 wt (SEQ ID NO:1), peptide DL03IL (SEQID NO:2), peptide DL03KP (SEQ ID NO:3), or peptide DL03LA (SEQ ID NO:4).Suitable fragments include CLIC1s that are truncated at the N- and/orC-terminus by one or more amino acids, typically by about 1 to 10-20amino acids, although fragments truncated by more amino acids may beused, provided the fragments bind an active DL03 compound. Additionally,mutants and/or fragments of CLIC1 that substantially retain one or moreof the biological activities of CLIC1 are useful in the assays and kitsof the invention. By “substantially retain” is meant that the mutant orfragment has at least 10% of the biological activity of CLIC1 asmeasured by any conventional assay of CLIC1 activity; preferably, themutant or fragment has at least 50% of the biological activity of CLIC1.

CLIC1 mutants useful in the methods and kits of the invention includeconservative mutants in which one or more amino acids is replaced withanother amino acid of the same class, as defined above in connectionwith the description of the DL03 compounds. Of course CLIC1 mutantsincluding non-conservative substitutions may also be used, so long asthe particular mutant binds an active DL03 compound and/or substantiallyretains CLIC1 activity. Thus, unless indicated otherwise, the expression“chloride intracellular channel 1” or “CLIC1” as used hereinspecifically includes such mutants and/or fragments in addition to thefull-length wild-type proteins.

The CLIC1s may be obtained using conventional recombinant andpurification techniques or may be isolated directly from the naturalsource. For example, any of the recombinant techniques discussed suprain connection with the DL03 compounds may be used to produce a CLIC1suitable for use in methods and kits of the invention. Suchrecombinantly-produced CLIC1s may be isolated using affinitychromatography (for example with an anti-CLIC1 antibody or otherconventional techniques. Specific examples that may be routinely adaptedare described in Tulk et al., Am J Physiol Cell Physiol. (2002)282(5):C1103-12; Harrop et al., 2001, supra; Tonini et al., FASEB J.2000 June; 14(9):1171-8; Tulk et al., J Biol Chem. (2000) September 1;275(35):26986-93, the disclosures of which are incorporated herein byreference. Other techniques for obtaining CLIC1s for use in the methodsand kits of the invention will be apparent to those of skill in the art.

Any screening technique known in the art for determining whethercompounds bind one another can be used to screen for compounds that binda CLIC1. The compounds screened can range from small organic moleculesto large polymers and biopolymers, and can include, by way of exampleand not limitation, small organic compounds, saccharides, carbohydrates,polysaccharides, lectins, peptides and analogs thereof, polypeptides,proteins, antibodies, oligonucleotides, polynucleotides, nucleic acids,etc. In one embodiment, the candidate compounds screened are smallorganic molecules having a molecular weight in the range of about100-2500 daltons. Such candidate molecules will often comprise cyclicalstructures composed of carbon atoms or mixtures of carbon atoms and oneor more heteroatoms and/or aromatic, polyaromatic, heteroaromatic and/orpolyaromatic structures. The candidate agents may include a wide varietyof functional group substituents. In one embodiment, the substituent(s)are independently selected from the group of substituents known tointeract with proteins, such as, for example, amine, carbonyl, hydroxyland carboxyl groups.

The candidate compounds may be screened on a compound-by-compound basisor, alternatively, using one of the myriad library techniques commonlyemployed in the art. For example, synthetic combinatorial compoundlibraries, natural products libraries and/or peptide libraries may bescreened using the assays of the invention to identify compounds thatbind a CLIC1. The candidate compounds may be assessed for the ability tobind a CLIC1 per se, or they may be assessed for the ability tocompetitively bind a CLIC1 in the presence of an active DL03 compound ofthe invention, such as peptide DL03 wt (SEQ ID NO:1), peptide DL03IL(SEQ ID NO:2), peptide DL03KP (SEQ ID NO:3), or peptide DL03LA (SEQ IDNO:4), or another compound that competitively binds a CLIC1 in thepresence of an active DL03 compound of the invention. These competitivebinding assays can identify compounds that bind the CLIC1 atapproximately the same site as the active DL03 compound. Myriadtechniques for carrying out competitive binding assays are known in theart. Any of these techniques may be employed in the present invention.

Such binding experiments may be conducted wholly in solution or,alternatively, either the CLIC1 or the candidate compound may beimmobilized on a solid support. For example, the CLIC1 or the candidatecompound may be attached to a glass or other bead or a solid surfacesuch as, for example, the bottom of a petri dish. The immobilization maybe mediated by non-covalent interactions or by covalent interactions.Methods for immobilizing myriad types of compounds and proteins on solidsupports are well-known. Any of these methods may be used to immobilizethe CLIC1 and/or candidate compound on solid supports.

The binding assays may employ a purified CLIC1 or, alternatively, theassays may be carried out with nucleosol and/or cytosol fractions fromcells that express the CLIC1, either endogenously or recombinantly.

Whether carried out in solution or with an immobilized CLIC1 orcandidate compound, the CLIC1 and candidate compound are typicallycontacted with one another under conditions conducive to binding.Although the actual conditions used can vary, typically the bindingassays are carried out under physiological conditions. Theconcentrations of CLIC1 and candidate compound used will depend upon,among other factors, whether the CLIC1 or candidate compound isimmobilized or free in solution, the binding affinities of candidatecompounds, etc. Actual concentrations suitable for a particular assaywill be apparent to those of skill in the art.

In many embodiments of the kits and assays of the invention it may beconvenient to employ a labeled CLIC1 and/or labeled candidate compound.For example, in one convenient embodiment, binding is assessed bycontacting an immobilized candidate compound with a labeled CLIC1 andassaying for the presence of immobilized label. For such embodiments,the label may be a direct label, i.e., a label that itself is detectableor produces a detectable signal, or it may be an indirect label, i.e., alabel that is detectable or produces a detectable signal in the presenceof another compound. The method of detection will depend upon thelabeled used, and will be apparent to those of skill in the art.

Examples of suitable direct labels include radiolabels, fluorophores,chromophores, chelating agents, particles, chemiluminescent agents andthe like. Suitable radiolabels include, by way of example and notlimitation, ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹Iand ¹⁸⁶Re. A suitable method for carrying out binding assays with achloride intracellular channel or biologically active fragments thereofis described in U.S. Pat. No. 6,228,616B1, the disclosure of which isincorporated herein by reference. Suitable fluorophores include, by wayof example and not limitation, fluorescein, rhodamine, phycoerythrin,Texas red, free or chelated lanthanide series salts such as Eu³⁺ and themyriad fluorophores available from Molecular Probes Inc., Eugene, Oreg.Examples of suitable colored labels include, by way of example and notlimitation, metallic sol particles, for example, gold sol particles suchas those described by Leuvering (U.S. Pat. No. 4,313,734); dye soleparticles such as described by Gribnau et al. (U.S. Pat. No. 4,373,932)and May et al. (WO 88/08534); dyed latex such as those described Snyder(EP 0 280 559 and 0 281 327) and dyes encapsulated in liposomes asdescribed by Campbell et al. (U.S. Pat. No. 4,703,017). Other directlabels that may be used will be apparent to those of skill in the art.

Examples of suitable indirect labels include enzymes capable of reactingwith or interacting with a substrate to produce a detectable signal(such as those used in ELISA and EMIT immunoassays), ligands capable ofbinding a labeled moiety, and the like. Suitable enzymes useful asindirect labels include, by way of example and not limitation, alkalinephosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphatedehydrogenase, lactate dehydrogenase and urease. The use of theseenzymes in ELISA and EMIT immunoassays is described in detail inEngvall, 1980, Methods in Enzymology, 70:419-439 and U.S. Pat. No.4,857,453.

Methods of labeling proteins and compounds with a variety of labels suchas those described above are well-known. Any of these methods may beused to label CLIC1s and/or candidate compounds. For example, a CLIC1may be labeled with a fluorophore such as fluorescein by incubating theCLIC1 with, for example, fluorescein isothiocyanate, using conventionaltechniques. Alternatively, a CLIC1 (or a candidate compound produced byrecombinant techniques) can be labeled metabolically by culturing cellsthat express the CLIC1 in the presence of culture medium supplementedwith a metabolic label, such as, by way of example and not limitation,[³⁵S]-methionine, one or more [¹⁴C]-labeled amino acids, one or more[¹⁵N]-labeled amino acids and/or [³H]-labeled amino acids (with thetritium substituted at non-labile positions).

In one embodiment of the invention, candidate compounds may be screenedfor the ability to bind a CLIC1 using an affinity chromatographytechnique. For example, a CLIC1 may be attached to a chromatographyresin according to standard techniques to create a CLIC1 affinity resinand this CLIC1 affinity resin used to identify compounds that bind theresin. Alternatively, the candidate compound could be bound to the resinand the resin used to determine whether it binds a CLIC1. In anotheralternative embodiment, an active DL03 compound of the invention may byattached to the chromatography resin. This DL03 affinity resin may thenbe used to bind a CLIC1 and the bound complex used to identify compoundsthat compete for binding the CLIC1 with the active DL03 compound,typically by washing the resin with a candidate compound and determiningwhether the candidate compound disrupts the CLIC1-DL03 compound complexby assaying for the release of CLIC1 from the resin.

Although candidate compounds may be screened for the ability to bind aCLIC1 on a compound-by-compound basis, it may be more convenient toscreen large numbers of candidate compounds simultaneously using one ofthe many library screening methodologies known in the art. One art-knownapproach uses recombinant bacteriophage to produce large libraries ofpeptides which can then be screened in a variety of formats for bindingto a CLIC1. Using such phage methods, very large libraries of candidatepeptides can be constructed (e.g., 10⁶-10⁸ peptides) and screened forbinding with a CLIC1. Methods for constructing and screening such “phagedisplay” libraries are described, for example, in Scott & Smith, 1990,Science 249:386-390; Cwirla et al., 1990, Proc. Natl. Acad. Sci.87:6378-6382; 1990); Devlin et al., 1990, Science 249:404-406 (1990);U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,432,018; U.S. Pat. No.5,580,717 and U.S. Pat. No. 5,723,286, the disclosures of which areincorporated herein by reference. Other non-limiting examples ofrecombinant library methodologies that may be used in connection withthe assays of the invention are described in U.S. Pat. No. 6,156,571;U.S. Pat. No. 6,107,059 and U.S. Pat. No. 5,733,731, the disclosures ofwhich are incorporated herein by reference.

A second art-known approach uses chemical methods to synthesizelibraries of compounds, such as small organic compounds, peptides and/orpeptide analogs, attached to beads or wafers that can then beconveniently screened for binding with a CLIC1. The libraries may beencoded or non-encoded. Methods of synthesizing such immobilizedlibraries, as well as methods of screening the libraries are described,for example, in Houghten, 1985, Proc. Natl. Acad. Sci. USA 82:5131-5735;Geysen et al., 1986, Molecular Immunology 23:709-715; Geysen et al.,1987, J. Immunologic Method 102:259-274; Frank & Döring, 1988,Tetrahedron 44:6031-6040; Fodor et al., 1991, Science 251:767-773; Furkaet al., 1988, 4th International Congress of Biochemistry, Volume 5,Abstract FR:013; Furka, 1991, Int. J. Peptide Protein Res. 37:487-493;Frank, 1992, Tetrahedron 48:9217-9232; Needels et al., 1993, Proc. Natl.Acad. Sci. USA 90:10700-10704; DeWitt et al., 1993, Proc. Natl. Acad.Sci. USA 90:6909-6913; Frank et al., 1993, Biorg. Med. Chem. Lett.3:425-430; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA90:10922-10926; WO 92/00252; WO 9428028; U.S. Pat. No. 6,329,143; U.S.Pat. No. 6,291,183; U.S. Pat. No. 5,885,837; U.S. Pat. No. 5,424,186;U.S. Pat. No. 5,384,261; U.S. Pat. No. 6,165,717; U.S. Pat. No.6,143,497; U.S. Pat. No. 6,140,493; U.S. Pat. No. 5,789,162; U.S. Pat.No. 5,770,358; U.S. Pat. No. 5,708,153; U.S. Pat. No. 5,639,603; U.S.Pat. No. 5,541,061; U.S. Pat. No. 5,525,735; U.S. Pat. No. 5,525,734;U.S. Pat. No. 6,261,776; U.S. Pat. No. 6,239,273; U.S. Pat. No.5,846,839; U.S. Pat. No. 5,770,455; U.S. Pat. No. 5,770,157; U.S. Pat.No. 5,609,826; U.S. Pat. No. 6,001,579; U.S. Pat. No. 5,968,736; U.S.Pat. No. 5,962,337; U.S. Pat. No. 5,789,172; U.S. Pat. No. 5,721,099;U.S. Pat. No. 5,565,324; U.S. Pat. No. 5,010,175; and U.S. Pat. No.4,631,211, the disclosures of which are incorporated herein byreference. For reviews of some of these techniques, see Ellman et al.,1996, Account, Chem. Res. 29:132-143; Gallop et al., 1994, J. Med. Chem.37:1233-1251; Gordon et al., 1994, J. Med. Chem. 37:1385-1401.Non-limiting examples of solid-phase chemical synthesis strategies andconditions useful for synthesizing combinatorial libraries of smallorganic and other compounds may be found in Bunin, 1998, TheCombinatorial Index, Academic Press, London, England (see, e.g., Chapter1-5) and Hermikens et al., 1996, Tetrahedron 52:4527-4554, as well asthe references cited therein, the disclosures of which are incorporatedherein by reference.

Another art-known approach utilizes solution-phase chemical synthesistechniques to synthesize libraries of compounds, such as, for example,libraries of small organic compounds, which may then be screened in theassays of the invention. Methods for synthesizing and screening suchsolution-phase libraries are well-known and are described, for example,in Bunin, 1998, The Combinatorial Index, Academic Press, England (see,e.g., Chapter 6); WO 95/02566; U.S. Pat. No. 5,962,736; U.S. Pat. No.5,766,481; U.S. Pat. No. 5,736,412 and U.S. Pat. No. 5,712,171, and thereferences cited therein; the disclosures of which are incorporatedherein by reference. Additional review articles, references, patents andbooks describing myriad techniques for synthesizing and screeninglibraries of compounds for the ability to bind another compound such asa CLIC1 can be found at Lebl & Leblova: Dynamic Database of Referencesin Molecular Diversity, Internet http://www.5z.com (see especially thediversity information pages at http://www.5z.com/divinfo).

Once a candidate compound that binds the CLIC1 has been identified,further assays may be carried out to characterize the bindingcharacteristics of the compound, for example, to determine its bindingaffinity, dissociation constant (Kd), on- and/or off-rates, etc., usingwell-known techniques. For example, binding affinities can be determinedusing saturation kinetics and Scatchard analysis. For saturationkinetics, the binding assay can be performed with increasingconcentrations of the candidate compound, which is typically labeledwith, for example, a radiolabel. Competitive binding experiments with anactive DL03 or other active compounds, for example peptide DL03 wt (SEQID NO:1), peptide DL03IL (SEQ ID NO:2), peptide DL03KP (SEQ ID NO:3), orpeptide DL03LA (SEQ ID NO:4), can be carried out with increasingconcentrations of unlabeled candidate compound and a fixed concentrationof labeled (for example radiolabeled) active DL03 or other compounds.

An alternative method for characterizing receptor/ligand bindingcharacteristics of a plurality of compounds in parallel that may beadapted for use in connection with the invention is described in U.S.Pat. No. 5,324,633.

In one embodiment of the invention, the candidate compounds identifiedwill have a dissociation constant (Kd) for CLIC1 on the order of 1 mM,100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM or even lower. In anotherembodiment of the invention, the candidate compounds identified willexhibit an IC₅₀ in a competitive binding assay with an active DL03compound of the invention or another compound that competes for bindinga CLIC1 with an active DL03 compound of the invention on the order of 1mM, 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM or even lower. In thiscontext, the IC₅₀ represents the concentration of candidate compoundthat displaces 50% of the bound DL03 or other compound. Suitable assaysfor measuring such IC₅₀s are well-known.

If desired, the ability of identified candidate compounds to modulate orregulate IL-4 induced IgE production and/or processes associatedtherewith can be confirmed in in vitro assays, such as those describedherein in connection with the identification of the DL03 compounds forexample as described in the Examples. Other assays that are well knownin the art may be used, for example, those described in U.S. Pat. No.5,958,707.

Knowledge of the interaction surface between a DL03 compound of theinvention such as peptide DL03 wt (SEQ ID NO:1), peptide DL03IL (SEQ IDNO:2), peptide DL03KP (SEQ ID NO:3) and/or DL03LA (SEQ ID NO:4) and aCLIC1, and in particular the CLIC1 amino acids involved in binding theDL03 compound, can also provide useful information for identifyingcompounds that bind a CLIC1. Identification and screening of CLIC1binding compounds is further facilitated by determining structuralfeatures of a CLIC1-DL03 compound complex, e.g., using X-raycrystallography, neutron diffraction, nuclear magnetic resonancespectrometry or any other techniques for structure determination. Thesetechniques provide for the rational design or in silico identificationof compounds that bind a CLIC1. The crystal structure of a Soluble formof hCLIC1 has been determined to 1.4-Å resolution. (Harrop et al.(2001), supra; PDB accession code 1KOM)

Candidate compounds identified using the screening assays of theinvention may be agonists or antagonists of the CLIC1. Thus, theidentified compounds may bind to the CLIC1 without activating it or mayinhibit one or more biological activities of CLIC1 (antagonist) or,alternatively, the identified compounds may activate one or morebiological activities of the CLIC1 (agonists). These functional assaysalso provide an indirect means of assessing whether a candidate compoundbinds a CLIC1. Observation of agonist or antagonist activity indicatesthe candidate compound binds the CLIC1. Thus, the method of theinvention may employ functional assays of CLIC1 activity to determinewhether a candidate compound binds CLIC1. Suitable methods fordetermining the effect of the candidate compound on the activity ofCLIC1 are well known and include, for example, methods described inHarrop et al. (2000, supra), Tulk et al. (2002, supra), Tulk et al.(2000, supra), Tonini et al. (2000, supra), Valenzuela et al. (2000,supra) and U.S. Pat. No. 5,854,411.

Cell-based functional assays for CLIC1 targets may be carried out inliving cells that express CLIC, either endogenously or recombinantly.For example, suitable methods include electrophysiological and membranepotential assays such as patch clamping, fast and slow response dyes,fluorescence resonance energy transfer (FRET), ion tracer assays, etc.).For reviews of these techniques, see Mattheakis et al., Curr Opin DrugDiscov Devel. 2001; Gonzalez et al., Drug Discov Today. 1999 September;4(9):431-439. January; 4(1):124-34., Xu et al., Drug Discov Today. 2001Dec. 15; 6(24):1278-1287, as well as the references cited therein, thedisclosures of which are incorporated herein by reference.

Kits

The invention also provides kits for carrying out the various screeningassays and methods of the invention. Such kits will typically include aCLIC1 and a compound that competes for binding with the CLIC1, such asan active DL03 compound. The CLIC1 and/or compound may be labeled orunlabeled. The kit may further include additional components useful forcarrying out the assays and methods. Non-limiting examples of suchadditional components include labels, labeling reagents, bindingbuffers, etc. The kit may also include instructions teaching its methodsof use. In one embodiment, the kit comprises and CLIC1 and a compoundselected from peptide DL03 wt (SEQ ID NO:1), peptide DL03IL (SEQ IDNO:2), peptide DL03KP (SEQ ID NO:3), peptide DL03LA (SEQ ID NO:4)peptide DL03TS (SEQ ID NO:5), peptide DL03GA (SEQ ID NO:6), peptideDL03TT (SEQ ID NO:7), peptide DL03AS (SEQ ID NO:8), peptide DL03MT (SEQID NO:9) and an analog thereof.

Uses of the DL03 Compounds and Identified Compounds

As discussed previously, the active DL03 compounds of the inventionand/or the active CLIC1-binding compounds identified by theabove-described screening methods (referred to collectively as “activecompounds”), can be used in a variety of in vitro, in vivo and ex vivoapplications to regulate or modulate processes involved with theproduction and/or accumulation of IgE. For example, the active compoundscan be used to modulate, and in particular inhibit, any or all of thefollowing processes in vitro, in vivo or ex vivo: IgE production and/oraccumulation; the IL-4 receptor-mediated signaling cascade leading toisotype switching and/or production of IgE; IL-4 induced switching ofB-cells to produce IgE, IL-4 mediated IgE production; and IL-4 inducedgermline ε transcription. In a specific embodiment of the invention, theactive compounds may be used to treat or prevent diseases characterizedby, caused by or associated with production and/or accumulation of IgE.Such treatments may be administered to animals in veterinary contexts orto humans. Diseases that are characterized by, caused by or associatedwith IgE production and/or accumulation, and that can therefore betreated or prevented with the active compounds include, by way ofexample and not limitation, anaphylactic hypersensitivity or allergicreactions and/or symptoms associated with such reactions, allergicrhinitis, allergic conjunctivitis, systemic mastocytosis, hyper IgEsyndrome, IgE gammopathies, atopic disorders such as atopic dermatitis,atopic eczema and/or atopic asthma, and B-cell lymphoma.

When used to treat or prevent such diseases, the active compounds may beadministered singly, as mixtures of one or more active compounds or inmixture or combination with other agents useful for treating suchdiseases and/or symptoms associated with such diseases. The activecompounds may also be administered in mixture or in combination withagents useful to treat other disorders or maladies, such as steroids,membrane stabilizers, 5LO inhibitors, leukotriene synthesis and receptorinhibitors, IgE receptor inhibitors, β-agonists, tryptase inhibitors andantihistamines, to name a few. The active compounds may be administeredper se or as pharmaceutical compositions.

Pharmaceutical compositions comprising the active compounds of theinvention may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making levigating, emulsifying,encapsulating, entrapping or lyophilization processes. The compositionsmay be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically. The actual pharmaceuticalcomposition administered will depend upon the mode of administration.Virtually any mode of administration may be used, including, for exampletopical, oral, systemic, inhalation, injection, transdermal, etc.

The active compound may be formulated in the pharmaceutical compositionsper se, or in the form of a pharmaceutically acceptable salt. As usedherein, the expression “pharmaceutically acceptable salt” means thosesalts which retain substantially the biological effectiveness andproperties of the active compound and which is not biologically orotherwise undesirable. Such salts may be prepared from inorganic andorganic acids and bases, as is well-known in the art. Typically, suchsalts are more soluble in aqueous solutions than the corresponding freeacids and bases.

For topical administration, the active compound(s) may be formulated assolutions, gels, ointments, creams, suspensions, etc. as are well-knownin the art.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives.

Alternatively, the injectable formulation may be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, etc., before use.To this end, the active compound(s) may dried by any art-knowntechnique, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art with, forexample, sugars or enteric coatings.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For rectal and vaginal routes of administration, the active compound(s)may be formulated as solutions (for retention enemas) suppositories orointments containing conventional suppository bases such as cocoa butteror other glycerides.

For administration by inhalation, the active compound(s) can beconveniently delivered in the form of an aerosol spray from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

For prolonged delivery, the active compound(s) can be formulated as adepot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt.

Alternatively, transdermal delivery systems manufactured as an adhesivedisc or patch which slowly releases the active compound(s) forpercutaneous absorption may be used. To this end, permeation enhancersmay be used to facilitate transdermal penetration of the activecompound(s). Suitable transdermal patches are described in for example,U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No.5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat.No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S.Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110;and U.S. Pat. No. 4,921,475.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well-known examples of delivery vehiclesthat may be used to deliver active compounds(s). Certain organicsolvents such as dimethylsulfoxide (DMSO) may also be employed, althoughusually at the cost of greater toxicity.

The pharmaceutical compositions may, if desired, be presented in a packor dispenser device which may contain one or more unit dosage formscontaining the active compound(s). The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

Gene Therapy

As will be recognized by skilled artisans, active compound(s) that arepeptides composed wholly of genetically-encoded amino acids may beadministered utilizing well-known gene therapy techniques. According tosuch techniques, a gene encoding the active compound may be introducedeither in vivo, ex vivo, or in vitro in a viral vector. Such vectorsinclude an attenuated or defective DNA virus, such as but not limitedto, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus(EBV), adenovirus, adeno-associated virus (AAV), and the like. Defectiveviruses, which entirely or almost entirely lack viral genes, arepreferred.

Defective virus is not infective after introduction into a cell. Use ofdefective viral vectors allows for administration to cells in aspecific, localized area, without concern that the vector can infectother cells. For example, in the treatment of the various diseasesdescribed herein, lymphocyte B-cells can be specifically targeted.Examples of particular vectors include, but are not limited to, adefective herpes virus I (HSV1) vector (Kaplitt et al., 1991, Molec.Cell. Neurosci. 2:320-330), an attenuated adenovirus vector, such as thevector described by Stratford-Perricaudet et al., 1992, J. Clin. Invest.90:626-630 and a defective adeno-associated virus vector (Samulski etal., 1987, J. Virol. 61:3096-3101; Samulski et al., 1989, J. Virol.63:3822-3828).

Preferably, for in vitro administration, an appropriateimmunosuppressive treatment is employed in conjunction with the viralvector, e.g., adenovirus vector, to avoid immuno-deactivation of theviral vector and transfected cells. For example, immunosuppressivecytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), oranti-CD4 antibody, can be administered to block humoral or cellularimmune responses to the viral vectors (see, e.g., Wilson, 1995, Nat.Med. 1(9):887-889). In addition, it is advantageous to employ a viralvector that is engineered to express a minimal number of antigens.

In another embodiment the gene can be introduced in a retroviral vector,e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann etal., 1983, Cell 33:153; Temin et al., U.S. Pat. No. 4,650,764; Temin etal., U.S. Pat. No. 4,980,289; Markowitz et al., 1988, J. Virol. 62:1120(1988); Temin et al., U.S. Pat. No. 5,124,263; Dougherty et al., WO95/07358; and Kuo et al., 1993, Blood 82:845. Targeted gene delivery isdescribed in WO 95/28494.

Alternatively, the vector can be introduced by lipofection. For the pastdecade, there has been increasing use of liposomes for encapsulation andtransfection of nucleic acids in vitro. Synthetic cationic lipidsdesigned to limit the difficulties and dangers encountered with liposomemediated transfection can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Felgner et. al., 1987, Proc.Natl. Acad. Sci. USA 84:7413-7417; Mackey et al., 1988, Proc. Natl.Acad. Sci. USA 85:8027-8031). The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes (Felgner & Ringold, 1989,Science 337:387-388). The use of lipofection to introduce exogenousgenes into the specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular celltypes would be particularly advantageous in a tissue with cellularheterogeneity, such as pancreas, liver, kidney, and the brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting (see Mackey et al., 1988, supra). Targeted peptides, e.g.,hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

It is also possible to introduce the vector as a naked DNA plasmid.Naked DNA vectors for gene therapy can be introduced into the desiredhost cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu & Wu, 1988, J. Biol. Chem., 263:14621-14624; CanadianPatent Application No. 2,012,311).

Naked nucleic acids encoding the active compound may also be introducedusing the gene-activated matrices described, for example, in U.S. Pat.No. 5,962,427.

Effective Dosages

The active compound(s) of the invention, or compositions thereof, willgenerally be used in an amount effective to treat or prevent theparticular disease being treated. The compound(s) may be administeredtherapeutically to achieve therapeutic benefit or prophylactically toachieve prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treated,e.g., eradication or amelioration of the underlying allergy, atopicdermatitis, atopic eczema or atopic asthma, and/or eradication oramelioration of one or more of the symptoms associated with theunderlying disorder such that the patient reports an improvement infeeling or condition, notwithstanding that the patient may still beafflicted with the underlying disorder. For example, administration ofan active compound to a patient suffering from an allergy providestherapeutic benefit not only when the underlying allergic response iseradicated or ameliorated, but also when the patient reports a decreasein the severity or duration of the symptoms associated with the allergyfollowing exposure to the allergen. Therapeutic benefit also includeshalting or slowing the progression of the disease, regardless of whetherimprovement is realized.

For prophylactic administration, the active compound may be administeredto a patient at risk of developing a disorder characterized by, causedby or associated with IgE production and/or accumulation, such as thevarious disorders previously described. For example, if it is unknownwhether a patient is allergic to a particular drug, the active compoundmay be administered prior to administration of the drug to avoid orameliorate an allergic response to the drug. Alternatively, prophylacticadministration may be applied to avoid the onset of symptoms in apatient diagnosed with the underlying disorder. For example, an activecompound may be administered to an allergy sufferer prior to expectedexposure to the allergen. Active compounds may also be administeredprophylactically to healthy individuals who are repeatedly exposed toagents known to induce an IgE-related malady to prevent the onset of thedisorder. For example, an active compound may be administered to ahealthy individual who is repeatedly exposed to an allergen known toinduce allergies, such as latex allergy, in an effort to prevent theindividual from developing an allergy.

The amount of active compound(s) administered will depend upon a varietyof factors, including, for example, the particular indication beingtreated, the mode of administration, whether the desired benefit isprophylactic or therapeutic, the severity of the indication beingtreated and the age and weight of the patient, the bioavailability ofthe particular active compound, etc. Determination of an effectivedosage is well within the capabilities of those skilled in the art.

Initial dosages may be estimated initially from in vitro assays. Forexample, an initial dosage for use in animals may be formulated toachieve a circulating blood or serum concentration of active compoundthat inhibits about 25% or more of IL-4 induced IgE production, or aprocess associated therewith, such as germline ε transcription, asmeasured in an in vitro assay. Alternatively, an initial dosage for usein animals may be formulated to achieve a circulating blood or serumconcentration of active compound that is equal to or greater than theIC₅₀ as measured in a CLIC1 competitive binding assay with an activeDL03 compound of the invention, such as peptide DL03 wt (SEQ ID NO:1),peptide DL03IL (SEQ ID NO:2), peptide DL03KP (SEQ ID NO:3), or peptideDL03LA (SEQ ID NO:4). Calculating dosages to achieve such circulatingblood or serum concentrations taking into account the bioavailability ofthe particular active compound is well within the capabilities ofskilled artisans. For guidance, the reader is referred to Fingl &Woodbury, “General Principles,” In: The Pharmaceutical Basis ofTherapeutics, Chapter 1, pp. 1-46, 1975, and the references citedtherein.

Initial dosages can also be estimated from in vivo data, such as animalmodels. Animals models useful for testing the efficacy of compounds totreat or prevent diseases characterized by, caused by or associated withIgE production and/or accumulation are well-known in the art. Suitableanimal models of hypersensitivity or allergic reactions are described inFoster, 1995, Allergy 50(21Suppl):6-9, discussion 34-38 and Tumas etal., 2001, J. Allergy Clin. Immunol. 107(6): 1025-1033. Suitable animalmodels of allergic rhinitis are described in Szelenyi et al., 2000,Arzneimittelforschung 50(11):1037-42; Kawaguchi et al., 1994, Clin. Exp.Allergy 24(3):238-244 and Sugimoto et al., 2000, Immunopharmacology48(1):1-7. Suitable animal models of allergic conjunctivitis aredescribed in Carreras et al., 1993, Br. J. Opthalmol. 77(8):509-514;Saiga et al., 1992, Ophthalmic Res. 24(1):45-50; and Kunert et al.,2001, Invest. Opthalmol. Vis. Sci. 42(11):2483-2489. Suitable animalmodules of systemic mastocytosis are described in O'Keefe et al., 1987,J. Vet. Intern. Med. 1(2):75-80 and Bean-Knudsen et al., 1989, Vet.Pathol. 26(1):90-92. Suitable animal models of hyper IgE syndrome aredescribed in Claman et al., 1990, Clin. Immunol. Immunopathol.56(1):46-53. Suitable animal models of B-cell lymphoma are described inHough et al., 1998, Proc. Natl. Acad. Sci. USA 95:13853-13858 and Hakimet al., 1996, J. Immunol. 157(12):5503-5511. Suitable animal models ofatopic disorders such as atopic dermatitis, atopic eczema and atopicasthma are described in Chan et al., 2001, J. Invest. Dermatol.117(4):977-983 and Suto et al., 1999, Int. Arch. Allergy Immunol.120(Suppl 1):70-75. Ordinarily skilled artisans can routinely adapt suchinformation to determine dosages suitable for human administration.

Dosage amounts will typically be in the range of from about 1 mg/kg/dayto about 100 mg/kg/day, 200 mg/kg/day, 300 mg/kg/day, 400 mg/kg/day or500 mg/kg/day, but may be higher or lower, depending upon, among otherfactors, the activity of the active compound, its bioavailability, themode of administration and various factors discussed above. Dosageamount and interval may be adjusted individually to provide plasmalevels of the active compound(s) which are sufficient to maintaintherapeutic or prophylactic effect. In cases of local administration orselective uptake, such as local topical administration, the effectivelocal concentration of active compound(s) may not be related to plasmaconcentration. Skilled artisans will be able to optimize effective localdosages without undue experimentation.

The compound(s) may be administered once per day, a few or several timesper day, or even multiple times per day, depending upon, among otherthings, the indication being treated and the judgement of theprescribing physician.

Preferably, the active compound(s) will provide therapeutic orprophylactic benefit without causing substantial toxicity. Toxicity ofthe active compound(s) may be determined using standard pharmaceuticalprocedures. The dose ratio between toxic and therapeutic (orprophylactic) effect is the therapeutic index. Active compound(s) thatexhibit high therapeutic indices are preferred.

The invention having been described, the following examples are offeredby way of illustration and not limitation.

EXAMPLES Identification of Peptide DL03 wt from a Random Library ofPeptide 20-mers

Peptide DL03 wt (SEQ ID NO:1) was identified by screening a retrovirallibrary of random peptide 20-mers for the ability to inhibit IL-4induced germline ε transcription using the HBEGF2a/diphtheria dualreporter phenotypic screening system described in WO 01/31232. Toconstruct the random library, A5T4 reporter cells (described in moredetail below) were infected with an infectious retroviral library ofrandom peptide 20-mers (prepared as described in WO 97/27213; see alsoWO 01/34806 at page 39, line 36 through page 40, line 19). Theretroviral vector used includes a gene encoding blue fluorescent protein(BFP) fused upstream of the region encoding the random peptide via alinker region encoding an α-helical peptide linker (expression fusionproduct is referred to as “BFP-peptide”). Expression of the BFP-peptideproduct is controlled by a promoter sensitive to thetetracycline-regulated transactivator such that expression of theBFP-peptide is regulated by tetracycline (Tet) or doxycycline (Dox). SeeU.S. patent application Ser. No. 10/096,339, entitled “Methods andCompositions for Screening for Altered Cellular Phenotypes”, filed onMar. 8, 2002. The BFP reporter gene provides a rapid phenotypic assay todetermine whether cells were infected: infected cells expressBFP-peptide and fluoresce blue (phenotype BFP⁺), uninfected cells do notexpress BFP-peptide, and do not fluoresce blue (phenotype BFP⁻). Toreduce the number of stop codons, the region of the vector encoding therandom peptide was of the sequence (NNK)₂₀, where each N independentlyrepresents A, T, C or G and K represents T or G. The library was alsobiased to account for degeneracy in the genetic code.

The A5T4 reporter cell line was engineered from BJAB B-cells (Menezes etal., 1975, Biomedicine 22:276-284; Source: Yoshinobu Matsuo, PhD.,Fujisaki Cell Center, Hayashibara Biochemical Labs, Inc., 675-1Fujisaki, Okayama 702-8006, Japan) and includes a reporter gene encodingthe HBEGF2a/GFP dual function reporter positioned downstream of anengineered 600 base pair IL-4 responsive fragment of an ε promoter (FIG.1; see also WO 99/58663) such that ultimate expression of the dualfunction reporter is driven by the ε promoter. When expressed, the dualfunction reporter cleaves into two pieces, a heparin-binding epidermalgrowth factor-like growth factor (HBEGF) and a green fluorescent protein(GFP), via the self-cleaving 2a sequence (Donnelly et al., 2001, J. Gen.Viol. 82:1027-1041; Donnelly et al., 1997, J. Gen. Virol. 78:13-21;Mattion et al., 1996, J. Virol. 70:8124; Ryan et al., 1994, EMBO J13:928-33 Ryan et al., 1991, J. Gen. Virol. 72:2727; Hellen et al.,1989, Biochem. 28:9881; see also, WO 99/58663). In this reporter system,cells ectopically expressing HBEGF are capable of translocatingdiphtheria toxin (DT) into their cytoplasm, leading to rapid, acutecytotoxicity. Cells that do not express HBEGF are spared this fate andcontinue to survive even in the presence of high concentrations of DT.The A5T4 reporter cell line was further engineered to express thetetracycline-regulated transactivator (tTA), allowing for regulation ofpeptide library expression with tetracycline (Tet) or doxycycline (Dox).See U.S. patent application Ser. No. 10/096,339, the disclosure of whichis incorporated herein by reference. Thus, according to this dualphenotypic reporter system, unstimulated control cells expressing arandom peptide fluoresce blue (BFP⁺) in the absence of Tet or Dox. Inthe presence of Tet or Dox, the peptide is not made and the cells areBFP⁻. Following stimulation with IL-4, BFP⁺ cells expressing anon-inhibitory peptide fluoresce green and, in addition, are sensitiveto DT. Stimulated BFP⁺ cells expressing an inhibitory peptide do notfluoresce green and are not DT sensitive. The toxin-conditionalselection and Tet or Dox-controlled peptide expression features of theA5T4 screening line are illustrated in FIGS. 2A & 2B, respectively.

Following infection, the library was enriched for cells expressingpeptides that inhibit IL-4 induced ε transcription as generally outlinedin the top half of FIG. 3 and sorted by FACS into single cell clones.The clones were then screened as generally illustrated in the lower halfof FIG. 3. Briefly, for screening, each clone was divided into twopopulations and one population was treated with Dox (10 ng/ml). After 5days, both populations were stimulated with IL-4 (final conc. 60 U/mL;PeptroTech, Inc.) and, after 3 more days, both populations were analyzedby FACS to measure BFP and GFP fluorescence. FACS data were converted toa “reporter ratio”, which is defined for this purpose as the ratio ofthe geometric mean of GFP fluorescence of the +IL-4/+Dox to the+IL-4/−Dox populations. Cells expressing a peptide that inhibitsgermline ε transcription have a reporter ratio of ≧1.1. A reporter ratioof ≧1.2 is indicative of strong inhibition.

The sequences of peptides expressed by positive clones (reporter ratiosof ≧1.1) were obtained by RT-PCR amplification of the integratedpeptide-expressing sequences. In this experiment, of 2.4×10⁹ A5T4 cellsinfected, 218 positive clones were identified, 199 of which were unique.From this same experiment, 155 total clones with a reporter ratio of≧1.19 were identified, 136 of which were unique. Clone DL03, whichencodes peptide DL03 wt, was amongst the positive clones identified(clone DL03 had a reporter ratio of 1.32).

Clone DL03 Transfers its Phenotype Into Naïve Cells

The ability of peptide DL03 wt (SEQ ID NO:1) to inhibit germline εtranscription was confirmed in naïve cells. Briefly, Phoenix cells weretransfected with a retroviral vector encoding a BFP-DL03 wt (SEQ IDNO:1) peptide fusion as described in WO 99/58663 and WO 97/27213. NaïveA5T4 cells were infected with the resultant virions and grown for 3days. The infected cells were stimulated with IL-4 (60 U/mL) and, after3 days, the cells were assessed by FACS for BFP and GFP. The FACS dataare presented in FIG. 4. As illustrated in FIG. 4, there are twopopulations of cells: infected cells that express the BFP-peptide fusion(BFP⁺) and uninfected cells that do not (BFP⁻). The BFP fluorescencedata corresponding to the BFP⁺ and BFP⁻ populations are provided inPanel A. The GFP fluorescence data corresponding to the BFP⁺ and BFP⁻populations are presented in Panel B. The reporter ratio for thispurpose is determined as the geometric mean of the GFP fluorescence ofthe BFP⁻ population divided by the geometric mean of the GFPfluorescence of the BFP⁺ population as presented in panel C. Thereporter ratio for the DL03 in this re-infection assay was 1.50.

Peptide DL03 wt Inhibits Transcription of an Endogenous Germline εPromoter

The ability of peptide DL03 wt (SEQ ID NO:1) to inhibit transcription ofan endogenous germline ε promoter was confirmed using a TAQMAN® assay(Roche Molecular, Alameda, Calif.). Briefly, A5T4 cells were infectedwith retrovirus capable of expressing peptide DL03 wt (SEQ ID NO:1)(prepared as described above). The cells were sorted for BFP⁺ to selectfor infected cells. Infected cells were divided into two populations.One population was exposed to Dox (10 ng/ml). Both populations werestimulated with IL-4 (60 U/ml). After 3 days, the cells were pelletedand the pellets assayed for ε promoter transcription using a TAQMANassay performed as described in Applied Biosystems Protocol 4310299(available at http://www.appliedbiosystems.com). The primers and probe,which are specific for the transcription product driven by the A5T4endogenous ε promoter, were as follows (the probe was labeled at the5′-end with Fam and at the 3′-end with Tamra): (SEQ ID NO:12) ε forwardprimer: ATCCACAGGCACCAAATGGA (SEQ ID NO:13) ε reverse primer:GGAAGACGGATGGGCTCTG (SEQ ID NO:14) ε probe: ACCCGGCGCTTCAGCCTCCA

The measured endogenous ε inhibition ratio, defined as the ratio of therelative expression units (TAQMAN quantitative PCR of ε transcriptionproduct) of +IL-4/+Dox to +IL-4/−Dox cells, was 2.11 (average of 3values; p=0.0035), indicating that peptide DL03 wt (SEQ ID NO:1)strongly inhibits the endogenous germline ε promoter.

Peptide DL03 wt is Selective for the Germline ε Promoter

To demonstrate selectivity for the germline ε promoter, peptide DL03 wt(SEQ ID NO:1) was tested for inhibition of germline α transcription. Theassay was similar to that described in the immediately precedingsection, except that ST486 cells (ATCC # CRL-1647) engineered to expressthe tetracycline-regulated transactivator were infected and the infectedcells were stimulated with TGF-β (40 ng/ml; Peprotech). The primers andprobe, which are specific for the transcription product driven by theST486 endogenous α promoter, were as follows (the probe was labeled atthe 5′-end with Fam and at the 3′-end with Tamra): (SEQ ID NO:15)α forward primer: CAGCACTGCGGGCCC (SEQ ID NO:16) α reverse primer:TCAGCGGGAAGACCTTGG (SEQ ID NO:17) α probe: CCAGCAGCCTGACCAGCATCCC

The measured endogenous α inhibition ratio was 0.66 (average of 3 valuesp=0.3228), indicating that peptide DL03 wt (SEQ ID NO:1) does notinhibit transcription of the germline α promoter. These data confirmthat peptide DL03 wt (SEQ ID NO:1) is a selective inhibitor of germlineε transcription.

IgE Synthesis and ELISA Assay

This example describes an IgE synthesis mixed lymphocyte and ELISA assaythat may be used to assess the amount of IgE produced by cells such ashuman peripheral blood lymphocytes or other lymphatic cells in thepresence and absence of candidate compounds, such as DL03 compoundsand/or compounds identified in the screening assays of the invention.

IgE Synthesis Assay

(a) Materials

In the various protocols that follow below, the following materials areused:

Heparin (Sigma H3393, St. Louis, Mo.)

Histopaque 1077 tubes (Sigma A0561, St. Louis, Mo.)

Iscove's Modified Dulbeccos Medium (“IMDM); Sigma 13390, St. Louis, Mo.)

Bovine Serum Albumin (“BSA”; Sigma A9418, St. Louis, Mo.).

Fetal Bovine Serum (“FCS”; Sigma F7524, St. Louis, Mo.). (the serum isheat inactivated prior to use at 56° C. for 30 minutes, aliquoted andstored at −20° C.; “HI-FCS”)

Human Transferrin (Sigma T2252, St. Louis, Mo.)

Bovine Insulin (Sigma 11882, St. Louis, Mo.)

200 mM L-Glutamine (“L-Gln”; Sigma G7513, St. Louis, Mo.) (stored as 5ml aliquots at −20° C.)

Pen/Strep 10% solution (Sigma P0781, St. Louis, Mo.) (stored as 5 mlaliquots at −20° C.)

PBS Dulbeccos (Gibco BRL 14190-094, now Invitrogen, Carlsbad, Calif.)

DMSO (Sigma D2650, St. Louis, Mo.)

Recombinant Human IL-4 (R&D Systems 204-IL, Minneapolis, Minn.) (storedas a stock solution of 100,000 U/ml in culture medium at −20° C.)

96 well tissue culture plates (Costar 3595, Corning Inc., Life Sciences,Acton MA)

96 well dilution blocks (Porvair 219008, Shepperton, UK)

Culture Medium: supplement 500 ml IMDM with 0.5% BSA (2.5 g), 10% HI-FCS(50 ml), 25 mg human transferrin, 2.5 mg bovine insulin, 2 mM L-Gln (5ml) and 1% pen/strep (5 mL). Filter sterilize before use.

(b) Blood Collection

Make up a 1 mg/ml solution of heparin in sterile PBS and place 1 ml ineach sterile 50 ml centrifuge tube required. Collect 50 ml of venousblood from a healthy human volunteer per centrifuge tube.

(c) Lymphocyte Isolation

Lymphocytes are isolated from the blood according to the protocol below:

-   -   1. Dilute blood with an equal volume of PBS containing 2%        HI-FCS.    -   2. Add 20 ml diluted blood to each histopaque tube. The        histopaque tubes should be warmed to room temperature before        use. They can be left overnight at room temp in the dark and        then spun at 1000 rpm for 5 min to settle the contents before        use. Spin at 1000 g for 35 min at room temperature in a benchtop        centrifuge with the break set to off.    -   3. Draw off the upper plasma layer and discard.    -   4. Draw off the lymphocyte layer into a sterile centrifuge tube.        If there is clear definition between the bottom of the        lymphocyte layer and the top of the frit, remove only the        lymphocyte layer, not all the liquid above the frit.    -   5. Add 30 ml PBS-2% HI-FCS to each 10 ml of cell suspension and        spin at 1000 rpm for 10 min at room temp.    -   6. Discard the supernatant and resuspend each cell pellet in        5-10 ml PBS-2% HI-FCS. Transfer the suspensions to a single tube        and bring the volume to 40 ml with PBS-2% HI-FCS. Spin at 1000        rpm for 10 min at room temp.    -   7. Repeat Step 6.    -   8. Wash the cells once with 40 ml culture medium.    -   9. Discard the supernatant and resuspend the pellet in 10 ml or        less culture medium.    -   10. Count the cells (using a Neubauer haemocytometer, a Coulter        Max-M cell counter or other counter).    -   11. Resuspend the cells in culture medium to a concentration of        2×10⁶ cells/ml. The cells can be left at this stage until they        are needed for assay set up.

(d) Assay Set Up

The assay is carried out as follows:

1. Dissolve test compounds in DMSO to give a 10 mM stock solution. Ifnecessary, sonicate the stock solution to aid dissolution of thecompound.

2. Dilute each compound stock solution 1:20 with culture medium to yielda 500 μM solution in 5% DMSO. Serially dilute this 500 μM stock solution1:10 with culture medium several times to provide enough stock solutionsto test the compounds over a range of concentrations (e.g., from 1 nM to10 μM).

3. Just prior to use, prepare a stock solution of IL-4 (1000 U/ml inculture medium).

4. To test the compounds and prepare appropriate controls, add to theappropriate wells of a multiwell plate the following reagents in thefollowing amounts: Reagent +IL-4 control −IL-4 control Sample TestCompound — — 50 μl IL-4 50 μl — 50 μl Culture Medium — 50 μl — 0.5% DMSOin 50 μl 50 μl — Culture Medium Cells 150 μl 150 μl 150 μl Total Volume250 μl 250 μl 250 μl

5. Incubate the plates for 10-12 days at 37° C. in a CO₂ incubator (5%CO₂/95% O₂). Following incubation, spin the plates at 1000 rpm for 10min and store at −20° C. until ready for the ELISA detection assay thatfollows below.

ELISA Assay for Detection of IgE

(a) Materials

In the assay protocol that follows below, the following materials areused:

Nunc maxisorp ELISA plates (GIBCO BRL, now Invitrogen, Carlsbad, Calif.)

Murine anti human IgE (GEI clone) (Sigma, St. Louis, Mo.)

Phosphate buffered saline (“PBS”; Sigma. St. Louis, Mo.)

Bovine serum albumin (“BSA”; Sigma, St. Louis, Mo.)

Polyethylenesorbitan monolaurate (“TWEEN 20”; Sigma, St. Louis, Mo.)

OPD tablets (DAKO Corp., Carpenteria, Calif.)

Streptavidin biotinylated horseradish peroxidase complex (“StreptavidinHRP”; Amersham, Piscataway, N.J.)

Human Myeloma protein IgE (The Binding Site, Inc.; San Diego, Calif.)

Biotinylated anti human IgE (Vector Laboratories, Inc., Burlingame,Calif.)

Hydrogen peroxide (Sigma, St. Louis, Mo.)

Distilled water (“dH₂O”)

Buffer A: 0.1 M NaHCO₃, pH 9.6

Buffer B: 0.1% TWEEN 20 in PBS

Buffer C: 1% BSA in Buffer B

Stop Solution: 0.6M H₂SO₄

(a) Protocol

The assay which measures the amount of IgE synthesized by the variouscontrols and samples prepared above, is carried out as described in thefollowing protocol:

-   1) Coat Nunc maxisorp plates with 50 μl of murine anti human IgE    (1:2000 in Buffer A). Leave plates at 4° C. overnight. Plates can be    stored in this way for a maximum of 7 days.-   2) Wash plates 3× with Buffer.-   3) Block any unbound sites on the plate by adding 200 μl of    buffer C. Incubate the plates for at least 2 hours at room    temperature. If blocking overnight incubate at 4 C. Blocked plates    should only be kept for a maximum of 2 days.-   4) Wash plates 3× with Buffer B.-   5) Place 50 μl of the sample or standard IgE diluted in Buffer C to    each well. IgE standards are diluted to give a concentration range    of 100 ng ml-0 ng/ml. To make up the 100 ng/ml standard, carry out    the following dilutions of stock IgE (0.5 mg/ml) in buffer C: 1 in    50 to give a 10 μg/ml solution (a minimum of 10 μls must be    transferred from the stock) followed by a 1 in 10 to give 1 μg/ml    and then a further 1 in 10 to give 100 ng/ml. Double dilutions are    then carried out in buffer C to give the rest of the standard curve.    Carry out all dilutions in glass bottles (10 oz) and make up at    least 1 ml of each so that reasonable volumes are being transferred    (one ELISA plate requires 100 μls of each standard). For the pilot    ELISA the standard curve is added to each plate just after the    samples have been added. Set up one ELISA plate with 3-4 columns of    standards only. This will be used to determine development time at    protocol Step 11.

For the pilot ELISA incubate the plates for one-two hours at roomtemperature

For the full ELISA incubate the plates overnight at 4° C.

-   6) Wash plates 3× with Buffer B.-   7) Add 50 μl of biotinylated anti human IgE (1/500 dilution in    Buffer B) to all wells. Incubate the plate for 1 hour at room    temperature.-   8) Wash plates 3× with Buffer B-   9) Add 50 μl of streptavidin HRP to each well (1/800 dilution in    Buffer B). Incubate the plates for 45 mins to 1 hour at room    temperature. The plates must not be left for longer than 1 hour at    this stage.-   10) Wash plates 3× with Buffer B.-   11) Add 50 μl of substrate (4 OPD tablets per 12 ml dH₂O plus 5 μl    hydrogen peroxide per 12 ml) to each well and wait for the color to    develop (this usually occurs within 10 minutes). Determine the time    taken to give the required OD (1.5-2 for 100 ng/ml of standard IgE)    using a plate containing only a standard curve. Develop all test    plates for this amount of time and in batches of 5 plates. This is    especially important if there are a large number of plates.-   12) Quench the reaction by adding 50 μl Stop Solution.-   13) Read plates at 492 nm within 30 minutes of stopping the    reaction.    Peptide DL03 wt Mediates its Inhibitory Action by Binding CLIC1    Identification of Potential Binding Partners for Peptide DL03 wt

Potential binding partners for peptide DL03 wt (SEQ ID: 1) wereidentified in a β-galactosidase yeast two-hybrid (YTH) assay usingpeptide DL03 wt (SEQ ID: 1) as bait and a cDNA library constructed fromthe A5T4 reporter cell line as prey. Binding was assessed byβ-galactosidase quantification using BetaFluor (Novagen) as a substrate.A negative interaction control (no cDNA fused downstream of the GAL4activation domain sequence) was also run. A general outline of the YTHassay is illustrated in FIG. 5A. Following clustering, filtering toremove non-specific bait hits (e.g., GFP and BFP), singletons andclusters recognized by 10 or more cDNA baits (based upon historical YTHassays), and prioritization, 22 prey clones were identified as hits.

Potential targets identified in the YTH assay were reconfirmed asgenerally outlined in FIG. 6. The cDNA clones identified as hits in theYTH assay were purified and rescreened for interaction with the DL03 wt(SEQ ID NO:1) peptide in a YTH assay. The hit clones were also screenedin a control assay for interaction with the BFP using a vector (pGBKT7)that contained only the BFP and not the DL03 peptide. At this stage, 12putative target clones remained.

In order to further discriminate among the putative targets,functional/interaction profile analyses were carried out as described incopending application Ser. No. 10/095,659, entitled Methods ofIdentifying Protein Targets, filed Mar. 8, 2002.

Confirmation that Peptide DL03 wt Mediates its Inhibitory Action byBinding CLIC1

Identification of colony 3.2DL03_Y_AS_U_(—)101 as the binding partnerfor peptide DL03 wt (SEQ ID NO:1) was confirmed using the profilingmethod described in copending application Ser. No. 10/095,659, entitledMethods of Identifying Protein Targets, filed Mar. 8, 2002 thedisclosure of which is incorporated herein by reference. The functionalprofile was obtained using the A5T4 reporter cell line and theinteraction profiles were obtained using the YTH assay and compared forcorrespondence. The main concept underlying this profiling method isthat mutants will tend to act the same way in both the functional assayand an interaction assay with the target polypeptide of the wild-typepeptide. That is, a mutant that exhibits an increase in function (ascompared to the wild-type peptide) in the functional assay will exhibitan increase in interaction (as compared to the wild-type peptide) in aYTH assay with the target polypeptide of the wild-type peptide. Statedanother way, the target polypeptide will yield an interaction profilethat corresponds closely to the functional profile when comparedvisually or by other means.

A collection of DL03 mutants was generated by replacing double aminoacid residues in the sequence of DL03 wt (SEQ ID NO:1) with a differentamino acid, typically an alanine, as described in copending applicationSer. No. 10/095,659. The functional profiles for the mutants derivedfrom DL03 wt (SEQ ID NO:1) was obtained by constructing and screeningfor activity in the A5T4 reporter cell line in the manner describedabove. The activity of each mutant at the germline ε promoter isreflected in the reporter ratio as described above. The reporter ratioswere the functional values that were used to develop the functionalprofiles.

TABLE 2 depicts the amino acid sequences (the dual mutations areunderlined) of the mutants tested and the measured reporter ratios whenscreening in the A5T4 reporter line. TABLE 2 Reporter Name PeptideSequence Ratio SEQ ID NO DL03wt LYTSILLHGATTASMTKPLA 1.50 (SEQ ID NO:1)DL03IL LYTSAALHGATTASMTKPLA 1.16 (SEQ ID NO:2) DL03KPLYTSILLHGATTASMTAALA 1.77 (SEQ ID NO:3) DL03LA LYTSILLHGATTASMTKPAR 1.22(SEQ ID NO:4) DL03TS LYAAILLHGATTASMTKPLA 1.49 (SEQ ID NO:5) DL03GALYTSILLHARTTASMTKPLA 1.67 (SEQ ID NO:6) DL03TT LYTSILLHGAAAASMTKPLA 1.43(SEQ ID NO:7) DL03AS LYTSILLHGATTRAMTKPLA 1.57 (SEQ ID NO:8) DL03MTLYTSILLHGATTASAAKPLA 1.30 (SEQ ID NO:9)

The interaction of DL03 wt (SEQ ID NO:1) peptide and its mutants withthe clones identified as potential targets for the peptide DL03 wt (SEQID NO:1) was then quantified in a galactosidase YTH assay. The YTH assaywas performed in the manner described above. A general outline of thisYTH assay is illustrated in FIG. 5B. For each clone tested, aninteraction profile was developed by comparing the β-galactosidaseactivity of each mutant to that of the wild type peptide as describedabove.

Comparison of the interaction and functional profiles was performed bycategorizing the mutants of DL03 based on the functional and interactionassays. Based on the reporter ratio, described in panel C of FIG. 4,each mutant was categorized into one of four functional categories: (1)reduction of function (ROF); (2) loss of function (LOF); (3) increase offunction (IOF) or (4) functionally neutral (N) as compared to theactivity of the wild type peptide, here DL03 wt (SEQ ID NO:1). Asmentioned previously, cells expressing a peptide that inhibits germlineε transcription have reporter ratios of ≧1.1. Cell expressing a loss offunction (LOF) mutant have a reporter ratio of <1.11. An increase offunction mutant (IOF) shows a >50% increase in reporter ratio and areduction of function (ROF) of mutant shows a >50% decrease in reporterratio. Functionally neutral mutants have reporter ratios that fallwithin ±50% of that of the DL03 wt (SEQ ID NO:1). The % increase ordecrease in reporter ratio is calculated after subtracting 1.0 from theindividual ratios. Using these criteria, peptide DL03IL (SEQ ID NO:2)and peptide DL03LA (SEQ ID NO:4) were designated as ROF mutants, peptideDL03KP (SEQ ID NO:3) was designated as a IOF mutant, peptides DL03TS(SEQ ID NO:5), DL03GA (SEQ ID NO:6), DL03TT (SEQ ID NO:7), DL03AS (SEQID NO:8), and DL03MT (SEQ ID NO:9) were designated as neutral. No LOFmutant were found for the DL03 clone.

The interaction of these IOF and ROF mutants with different polypeptidesencoded by the putative target clones were quantified using the YTHassay described above. Based on the YTH assay, for each putative target,the mutants were categorized into the following four interactioncategories: (1) reduction of interaction (ROI); (2) loss of interaction(LOI); (3) increase of interaction (IOI); and (4) interactionallyneutral (N), by assessing the binding affinity ratio of the mutantpeptide and the wild type peptide (β-gal activity of mutant/wild type)to the putative target. For the YTH interaction assay mutants DL03IL(SEQ ID NO:2), DL03KP (SEQ ID NO:3), DL03LA (SEQ ID NO:4), as well asoriginally identified peptide DL03 wt (SEQ ID NO:1) were used. Theselection criteria for categorizing the interactions is shown in FIG. 8.

Based on these functional and interaction categorizations, graphicprofile representations for each potential target polypeptide wereobtained using a weighted categorization process as depicted in FIG. 9.In FIG. 9, the functional profile (reporter ratios of DL03 wt (SEQ IDNO:1) and its three mutants) is plotted along the X-axis and theinteraction profile (β-galactosidase signal from the YTH assay for DL03wt (SEQ ID NO:1) and its three mutants) is plotted along the Y-axis. TheX-axis is further categorized into L (loss of function), R (reduction offunction), N (functionally neutral), and I (increase of function) basedon the reporter ratios. A ratio of <1.11 is categorized as an L and Nincludes ratios that fall within ±50% of the wild type peptide ratio. Aratio that is greater than +50% of the wt peptide ratio is I. R includesratios greater than 1.11 and ratios less than −50% of the wt peptideratio. Similarly, the Y-axis is further categorized based on thecriteria in FIG. 8. In such a graphic profile, profiles that correspondclosely have interaction values (in this case, β-galactosidase signal)and functional values (in this case, reporter ratios) that fall along aline with a positive slope. Also, close correspondence is indicated whenthe mutants fall within the same category type using both the reporterratio and the β-galactosidase signal, i.e., a mutant categorized as a Lbased on the reporter ratio is also categorized as a L based on theβ-galactosidase signal. In this case, the interaction and functionalprofiles for clone 3.2DL3_Y_AS_U_(—)0101 fall along a line with apositive slope. Each of the three mutants also fall within the samerespective categories based on both reporter ratio and β-galactosidasesignal. The profiles for the other putative target clones selected inthe initial YTH screen do not satisfy these criteria.

Based on the close correspondence observed between the interaction andfunctional profiles, the polypeptide encoded by clone3.2DL3_Y_AS_U_(—)0101, was identified as a target for peptide DL03 wt(SEQ ID NO:1). The clone was sequenced and a sequence comparison (usinga Blast search with default parameters) with sequences in the NCBI(GenBank) nucleic database carried out. From this sequence comparisonthe clone 3.2DL3_Y_AS_U_(—)0101 was identified as human chlorideintracellular channel 1, (NCBI accession # NP_(—)001279.2;XP_(—)004183.3), also called p64CLCP.

While the invention has been described by reference to various specificembodiments, skilled artisans will recognize that numerous modificationsmay be made thereto without departing from the spirit and the scope ofthe appended claims.

All references cited throughout the disclosure are incorporated hereinby reference in their entireties for all purposes.

1. A method of identifying a compound that modulates IL-4receptor-mediated IgE production or a process associated therewith,comprising determining whether the compound binds a CLIC1, wherein theability to bind the CLIC1 identifies the compound as being a modulatorof IL-4 induced IgE production or a process associated therewith. 2-57.(canceled)